CN111407879A - 山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用 - Google Patents
山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用 Download PDFInfo
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Abstract
本发明涉及一种山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用,属于医药或健康产品领域。本发明从体内及体外研究系统中多层次、全方面证实了山药蛋白提取物对肾阳虚型实验模型勃起功能障碍中的改善作用,与勃起控制相关器官功能的改善密切相关,且其保护作用区别于5型磷酸二酯酶抑制剂。包含利用其为原料制备的具有改善和、或治疗勃起功能障碍作用的药品及健康产品,特别但不限于应用于改善和、或治疗哺乳动物勃起功能障碍。
Description
技术领域
本发明属于医药或健康产品领域,尤其涉及一种山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用,包含利用其为原料制备的具有改善和、或治疗勃起功能障碍作用的药品及健康产品,特别但不限于应用于改善和、或治疗哺乳动物勃起功能障碍。
背景技术
中医理论认为勃起功能障碍(erectile dysfunction,ED)属于“阳痿”、“不举”等范畴。治疗时强调以整体观念辨证论治,以肾为治病核心,其中肾阳虚证是ED的主要诱因。现代医学表明,肾阳虚证由体内多种代谢途径紊乱引起,进而导致ED的发生。
目前口服5型磷酸二酯酶(PDE5)抑制剂,如西地那非(Sildenafil),是一线的ED治疗药物。这些药物能特异性针对NO/cGMP通路中的PDE5,从而抑制cGMP水解,有利于维持海绵体平滑肌舒张,发挥器官功能。然而,对于严重内皮功能障碍及器官功能损伤的ED(如糖尿病性ED、高血压性ED及老年性ED等)一直束手无策。
中药具有整体调节、多靶点、副作用小等优势,目前多种滋补肾阳单味中药或复方制剂对于治疗ED均展现了极好的作用效果。山药为薯蓣科植物薯蓣(Dioscorea oppositaThunb.)的干燥根茎,是我国传统的健康食品和药食同源类中药,具有补脾养胃,生津益肺,补肾涩精的功效,用于脾虚食少,久泻不止,肺虚咳喘,肾虚遗精,带下,尿频,虚热消渴等症。已报到的山药中含有维生素、蛋白质、淀粉、游离氨基酸等多种营养成分,钙、磷和铁等一些矿物质的含量也极为丰富,其中报道的功能性活性成分包括多糖、多酚类化合物、皂苷、尿囊素、胆甾醇、麦角甾醇、胆碱等,具有抗氧化、抗肿瘤、降血脂、调节肠道菌群、增强机体免疫等多种生理作用。例如山药多糖具有增强体液免疫、抗氧化、抗肿瘤、调节胃肠道、降血糖等多种功能;多酚类物质是山药有效的抗氧化活性物质,可有效的清除体内自由基,具有降血脂功能,并对山药的感官和风味产生影响;山药中的尿囊素,可以改善肌肤,在皮肤和组织修复上有很好的作用;薯蓣皂苷分布于山药的根、茎、叶等各个部位,具有抗炎消肿、镇痛、抗氧化、降低心脑血管疾病、预防癌症、抗肿瘤、保护生殖系统等作用。关于山药提取物薯蓣皂苷保护生殖系统的报道显示,山药提取物薯蓣皂苷能缩短雷公藤多苷诱导的少弱精子症小鼠勃起潜伏期,提高精子质量及生殖器官、免疫器官的脏器系数,并提高睾丸组织中SOD活力及降低MDA含量,文中没有对其作用机制进行后续研究报告。
关于山药中蛋白质类成分研究的报道,主要集中在蛋白提取方面,活性报道较少。已发表的活性研究报道,如体外抗氧化活性研究,人食道癌细胞EC-109的抑制作用研究,膜蛋白酶抑制剂活性研究,α-葡萄糖苷酶的体外抑制作用研究,提高免疫力研究,提高脑细胞线粒体氧化代谢酶活性的研究,以及在治疗肾炎及肾性高血压的药物中的用途,在以肥胖、胰岛素抵抗、高血压、高血脂、脂肪肝为特征的代谢综合征及其所致心脏和肾脏损害方面的应用等。
截至目前,国内现有文献未见对山药蛋白提取物(CYCSE)基于肾阳虚模型的勃起功能障碍应用方面的报道。山药对性功能障碍的改善作用模型建立及评价体系单一、物质基础尚不完全明确,且作用机制研究几近空白。
发明内容
本发明提供一种山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用,该山药蛋白提取物制备工艺安全、对人体无毒副作用,从体内及体外研究系统中多层次、全方面证实了山药蛋白提取物对肾阳虚型ED的改善作用,与勃起控制相关器官功能的改善密切相关,且其保护作用区别于PDE5抑制剂。本应用特别但不限于应用于改善和、或治疗哺乳动物勃起功能障碍。
本发明采取的技术方案是:
一种山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用。
本发明所述勃起功能障碍为肾阳虚导致的勃起功能障碍。
本发明包含利用其为原料制备的具有改善和、或治疗勃起功能障碍作用的药品及健康产品,特别但不限于应用于改善和、或治疗哺乳动物勃起功能障碍。
本发明肾阳虚模型是基于氢化可的松诱导的大鼠肾阳虚模型,从对海绵体组织形态的改善以及对海绵体平滑肌内皮细胞功能的修复和勃起关键信号途径(NO/cGMP)的激活,证明山药蛋白提取物对肾阳虚大鼠勃起功能障碍(erectile dysfunction,ED)治疗的有效性。
本发明山药蛋白提取物改善肾阳虚大鼠睾丸功能的作用是通过改善睾丸组织形态、减少睾丸功能细胞凋亡、增加睾丸间质细胞含量、促进睾酮分泌、增强精子活力及改善睾丸纤维化六个方面共同确定的。
本发明中的山药蛋白提取物(CYCSE)通过以下方式制备:取鲜山药用8-20倍量蒸馏水匀浆,静置1~4h,4~20℃,过滤,上清液调节pH至1~2,过滤,取沉淀,沉淀调节pH=7~8,冻干,此蛋白收率在0.5%~3%之间,蛋白呈疏松的白色至灰白色粉末,气微,味淡。
优选的是,取鲜山药用15倍量蒸馏水匀浆,静置2h,4℃,过滤,上清液用HCl调节pH=2.0,过滤,取沉淀,沉淀用NaOH调节pH=7.0,冻干。
本发明的山药蛋白提取物(CYCSE)可以添加常用的药用辅料制成药学上可接受的口服制剂如口服汤剂、片剂、胶囊剂或颗粒剂等,且所述口服汤剂、片剂、胶囊剂或颗粒剂均可采用相应种类制剂的常规制备方法来制得。
对于应用本发明的山药蛋白提取物(CYCSE)制备健康产品没有限制。因此,所述提取物可以添加在饮料、冲剂、米糕、巧克力、糖果、饼干、口香糖、茶、含酒精饮料以及复合维生素等。
本发明的有益效果:
(1)本发明应用山药蛋白提取物,开发了一项在勃起功能障碍方面的新应用。
(2)本发明对所制备的山药蛋白提取物的物质基础进行了分析,此山药蛋白提取物是包含36%蛋白质及62%淀粉,蛋白质的分子量分布是32kDa、14.4kDa。蛋白分子量较小,无异味,易于吸收,提取过程主要采用水为溶剂,对人体无毒副作用。
(3)本发明中山药蛋白提取物对肾阳虚ED的治疗效果进行了较为系统的研究。首先,基于氢化可的松诱导的大鼠肾阳虚模型,从对海绵体组织形态的改善以及对海绵体平滑肌内皮细胞功能的修复和勃起关键信号途径(NO/cGMP)的激活,明确了CYCSE对肾阳虚大鼠ED的有效性。又从改善睾丸形态、减少睾丸功能细胞凋亡、增加睾丸间质细胞含量、促进睾酮分泌、增强精子活力以及改善睾丸纤维化全面确证了CYCSE具有改善睾丸功能的作用,且其作用效果明显优于西地那非。
(4)本发明中结合体内及体外研究系统对CYCSE的作用机制进行了探索。证明CYCSE能够诱导Nrf2蛋白表达,激活Nrf2/HO-1信号途径,启动抗氧化防御系统,抵抗睾丸氧化应激;同时,能够通过激活TGF-β1/SMAD信号途径,改善睾丸纤维化,维系器官功能。最后,研究利用过氧化氢(H2O2)诱导TM3细胞及勃起功能控制细胞(原代阴茎海绵体平滑肌内皮细胞)产生氧化应激损伤,进一步验证了CYCSE对功能细胞损伤的恢复作用。在TM3细胞中,CYCSE能够通过激活ERK和AKT信号途径改善H2O2诱导的细胞活力降低、促进睾酮分泌及增加cGMP含量,能够通过Nrf2/HO-1信号途径激活抗氧化防御系统降低活性氧簇(ROS)聚集,通过TGF-β1/SMAD2/3信号通路改善细胞纤维化程度,且其保护作用明显强于西地那非。采用Matrigel 3D培养系统分离培养小鼠原代海绵体内皮细胞(MCECs),亦可证明CYCSE能够增加H2O2诱导的MCECs细胞活力,通过勃起关键通路AKT/eNOS/cGMP的级联反应发挥保护作用,而西地那非无作用效果。
附图说明
图1是CYCSE蛋白丰度分析图;
图2是CYCSE的蛋白分子量分布图;
图3是CYCSE对肾阳虚大鼠阴茎海绵体组织形态的影响-对照组图(放大倍数:200x);
图4是CYCSE对肾阳虚大鼠阴茎海绵体组织形态的影响-肾阳虚组图(放大倍数:200x);
图5是CYCSE对肾阳虚大鼠阴茎海绵体组织形态的影响-低浓度组图(放大倍数:200x);
图6是CYCSE对肾阳虚大鼠阴茎海绵体组织形态的影响-高浓度组图(放大倍数:200x);
图7是CYCSE对肾阳虚大鼠阴茎海绵体组织形态的影响-西地那非组图(放大倍数:200x);
图8是基于Matrigel 3D培养系统的MCECs分离培养方案图;
图9是CYCSE对MCECs细胞活力的影响图,***p<0.001vs.模型组;
图10是CYCSE对肾阳虚大鼠iNOS含量的影响图,###p<0.001vs.对照组;*p<0.05,**p<0.01,***p<0.001vs.模型组;
图11是CYCSE对肾阳虚大鼠cGMP含量的影响图,###p<0.001vs.对照组;***p<0.001vs.模型组;
图12是Western blot检测MCECs细胞p-AKT/AKT、p-eNOS/eNOS的表达情况图;
图13是定量分析MCECs细胞p-AKT/AKT表达量图,###p<0.001vs.模型组;*p<0.05,***p<0.001vs.模型组;
图14是定量分析MCECs细胞p-eNOS/eNOS表达量图,###p<0.001vs.模型组;**p<0.01,***p<0.001vs.模型组;
图15是CYCSE对MCECs细胞cGMP含量的影响图,###p<0.001vs.模型组;*p<0.05,***p<0.001vs.模型组;
图16是CYCSE对肾阳虚大鼠睾丸形态的影响-对照组图(放大倍数:300x);
图17是CYCSE对肾阳虚大鼠睾丸形态的影响-肾阳虚组图(放大倍数:300x);
图18是CYCSE对肾阳虚大鼠睾丸形态的影响-低浓度组图(放大倍数:300x);
图19是CYCSE对肾阳虚大鼠睾丸形态的影响-高浓度组图(放大倍数:300x);
图20是CYCSE对肾阳虚大鼠睾丸形态的影响-西地那非组图(放大倍数:300x);
图21是CYCSE对肾阳虚大鼠睾丸功能细胞凋亡的影响图,###p<0.001vs.对照组;***p<0.001vs.模型组;
图22是CYCSE对肾阳虚大鼠睾丸组织8-OHdG含量的影响图,###p<0.001vs.对照组;**p<0.01,***p<0.001vs.模型组;
图23是CYCSE对肾阳虚大鼠睾丸组织SOD水平的影响图,###p<0.001vs.对照组;*p<0.05,***p<0.001vs.模型组;
图24是CYCSE对TM3细胞ROS水平的影响图,###p<0.001vs.对照组;***p<0.001vs.模型组;
图25是Western blot检测肾阳虚大鼠睾丸组织Nrf2蛋白的表达情图;
图26是定量分析肾阳虚大鼠睾丸组织Nrf2蛋白表达量图,#p<0.05vs.对照组;**p<0.01vs.模型组;
图27是CYCSE对TM3细胞中Nrf2,NQO1的mRNA表达量的影响图,#p<0.05vs.对照组;**p<0.01vs.模型组;
图28是Western blot检测TM3细胞中Nrf2总蛋白、Nrf2胞浆蛋白、Nrf2核蛋白、HO-1蛋白的表达情况图;
图29是定量分析TM3细胞中Nrf2总蛋白、Nrf2胞浆蛋白、Nrf2核蛋白、HO-1蛋白的表达量图,###p<0.001vs.对照组;*p<0.05,***p<0.001vs.模型组;
图30是CYCSE对TM3细胞活力的影响图,###p<0.001vs.模型组;***p<0.001vs.CYCSE组。Sil:Sildenafil,西地那非;PD:ERK抑制剂PD98059;LY:AKT抑制剂LY294002;
图31是Western blot检测TM3细胞中p-ERK/ERK、p-AKT/AKT的表达情况图;
图32是定量分析TM3细胞中p-ERK/ERK的表达量图,###p<0.001vs.模型组;**p<0.01vs.CYCSE组。Sil:Sildenafil,西地那非;PD:ERK抑制剂PD98059;LY:AKT抑制剂LY294002;
图33是定量分析TM3细胞中p-AKT/AKT的表达量图,###p<0.001vs.模型组;**p<0.01,***p<0.001vs.CYCSE组。Sil:Sildenafil,西地那非;PD:ERK抑制剂PD98059;LY:AKT抑制剂LY294002;
图34是CYCSE对TM3细胞cGMP含量的影响图,###p<0.001vs.模型组;***p<0.001vs.模型组;
图35是CYCSE对肾阳虚大鼠睾酮含量的影响图,*p<0.05,***p<0.001vs.模型组;#p<0.001vs.CYCSE(80mg/kg);
图36是CYCSE对TM3细胞睾酮含量的影响图,##p<0.01vs.模型组;*p<0.05vs.CYCSE组;
图37是Western blot检测肾阳虚大鼠睾丸组织中TGF-β1/SMAD2/3信号通路的表达情况图;
图38是定量分析肾阳虚大鼠睾丸组织中TGF-β1/SMAD2/3信号通路的表达量图,###p<0.001vs.对照组;**p<0.01,***p<0.001vs.模型组;
图39是TM3细胞中TGF-β1荧光强度的量化分析图,###p<0.001vs.对照组;***p<0.001vs.模型组;
图40是Western blot检测TM3细胞中TGF-β1/SMAD2/3信号通路的表达情况图;
图41是定量分析TM3细胞中TGF-β1/SMAD2/3信号通路的表达量图,###p<0.001vs.对照组;***p<0.001vs.模型组。
具体实施方式
下面结合具体实施例对本发明做进一步详细的说明,但实施例并不对本发明做任何形式的限定,以下实施例中的方法、设备、材料,如果未特别说明,均为本领域常规的方法、设备和材料。
实施例1本发明一种山药蛋白提取物颗粒剂在勃起功能障碍中的应用。
鲜山药2.0kg,加入14倍量蒸馏水匀浆,静置3h,10℃,过滤,上清液用HCl调节pH至2,过滤,取沉淀,沉淀用NaOH调节pH=7.0,冻干,山药蛋白提取物收率2.0%。加入辅料(砂糖、淀粉、糊精、葡萄糖等),制成颗粒剂1000g。
实施例2本发明一种山药蛋白提取物口服液在勃起功能障碍中的应用。
鲜山药3.0kg,加入20倍量蒸馏水匀浆,静置1h,4℃,过滤,上清液用HCl调节pH=1,过滤,取沉淀,沉淀用NaOH调节pH=8.0,冻干,山药蛋白提取物收率1.8%。加入辅料(促纯净水,白砂糖,阿斯巴甜,黄原胶,CMC钠等),制成口服液1.5L。
实施例3本发明一种山药蛋白提取物在健康产品压片糖果中的应用。
鲜山药2.0kg,加入8倍量蒸馏水匀浆,静置4h,20℃,过滤,上清液用HCl调节pH=1.5,过滤,取沉淀,沉淀用NaOH调节pH=7.5,冻干,山药蛋白提取物收率2.1%。加入压片糖果辅料(白砂糖,淀粉,糊精,乳糖,硬脂酸镁、微晶纤维素、甘露醇等),制成压片糖果1.0kg,本压片糖果能明显的发挥山药补肾涩精的传统功效,适合肾虚人群食用。
实施例4山药蛋白提取物的制备
取鲜山药2.0kg,用15倍量蒸馏水匀浆,静置2h,4℃,过滤,上清液用HCl调节pH=2.0,过滤,取沉淀,沉淀用NaOH调节pH=7.0,冻干。
本发明中的山药蛋白提取物(以下简称CYCSE)在勃起功能障碍中的应用,具体药理实验和作用机制研究方法如下:
1.实验方法:
1.1实验细胞与动物
TM3细胞:购自中国科学院上海细胞库,培养条件为DMEM/F12+5%马血清+2.5%胎牛血清+1%双抗,37℃、5%CO2培养箱。
SD大鼠:购自亿斯实验动物技术有限公司,SPF级,180-200g。(合格证编号:SCSK(吉)2018-0007)
C57BL/6小鼠:购自亿斯实验动物技术有限公司,SPF级,22-24g。(合格证编号:SCSK(吉)2018-0007)
1.2山药蛋白提取物(CYCSE)成分分析
根据BCA蛋白检测试剂盒(碧云天)和淀粉含量检测试剂盒(索莱宝)的说明书,分别测定CYCSE中蛋白质和淀粉的含量。卡马斯亮蓝法检测CYCSE中的蛋白质,凝胶成像仪拍照并分析蛋白质分子量的分布,L-8900型氨基酸自动分析仪测定CYCSE氨基酸含量,酶标仪检测吸光度并计算淀粉含量。
1.3肾阳虚大鼠模型的构建
SD大鼠随机分成5组,每组10只。灌胃25mg/kg氢化可的松(HCT)10d构建肾阳虚模型,然后给予不同浓度CYCSE 10d。具体分组如下:对照组(蒸馏水20d);肾阳虚组(HCT 10d+蒸馏水10d);CYCSE低浓度组(HCT 10d+60mg/kg CYCSE 10d);CYCSE高浓度组(HCT 10d+80mg/kg CYCSE 10d);西地那非组(HCT 10d+4.4mg/kg西地那非10d)。治疗后,腹腔注射3%戊巴比妥钠麻醉大鼠。
1.4生化标记物的检测
动物麻醉后,腹腔动脉取血后自然凝固25min,4℃条件下2500rpm/min离心10min。采集上清液,即为血清样本。按大鼠ELISA检测试剂盒(朗顿)的说明书检测血清中诱导型一氧化氮合酶(iNOS)、环磷酸鸟苷(cGMP)、睾酮含量。
1.5精子数量及活力检查
在给药结束时,收集附睾样品,清除多余组织,生理盐水洗去血液。用镊子固定,纵向切割附睾尾侧,在含有PBS的培养皿中释放精子,在室温下放置10分钟,用血细胞计数器在显微镜下检测精子数量及活力(精子活力分为4个等级:a快速直线向前运动、b缓慢直线向前运动、c原地运动、d不能运动)。
1.6组织学检查
将海绵体组织和睾丸组织固定在4%多聚甲醛中,并进行石蜡包埋和切片处理。睾丸组织切片用苏木精-伊红法和Masson法分别染色。海绵体组织切片用苏木精-伊红法染色。免疫组化法处理睾丸组织切片,用3β-HSD抗体标记睾丸间质细胞。尼康体视显微镜拍摄照片。
1.7ELISA法检测睾丸组织氧化应激
用8-羟基-2-脱氧鸟苷(8-OHdG)和超氧化物歧化酶(SOD)活性评价氧化应激。将液氮中的睾丸组织研磨成粉末,于PBS中匀浆后2500rpm/min离心25min。收集上清液,按大鼠ELISA检测试剂盒说明书检测大鼠睾丸组织中8-OHdG含量和SOD活性。
1.8TUNEL染色
根据TUNEL原位细胞死亡检测试剂盒(罗氏)的说明书进行睾丸组织中细胞凋亡检测。细胞核用DAPI染色5min。荧光显微镜拍摄照片。
1.9原代海绵体内皮细胞(MCECs)的分离培养
取8周龄C57BL/6J小鼠脱颈处死,酒精棉球对其下腹部消毒,用镊子、手术剪做下腹部切口,剥开腹部筋膜及包皮腺,露出海绵体组织。用手术剪分离海绵体置于含10%双抗的Hank’s平衡盐溶液中,镜下除去尿道及神经血管束,获得干净的海绵体组织。在含10%双抗的PSB中清洗3次,利用精细手术剪剪成1-2mm3小块,置于预冷的24孔板底,每孔放置2块切块。每孔补加200μL含50ng/mL VEGF-A的Matrigel基质胶包被切块,模拟3D环境培养MCECs并诱导其增殖。于37℃、5%CO2培养箱中培养14d至长满孔底,吸除培养基,每孔加200μL Dispase酶于培养箱中消化1h,加入等体积10mM EDTA终止消化,离心后取细胞沉淀置于MCECs专用培养基中培养,2-3代进行后续实验。
1.10MCECs的纯度鉴定
利用免疫荧光法对MCECs的纯度进行鉴定。取密度为5*104cells/mL的MCECs在6孔板内做细胞爬片处理,贴壁24h后,吸除培养基,用预冷的PBS洗3次,加4%多聚甲醛室温固定15min,预冷的PBS洗3次。加含5%山羊血清的0.5%Triton X-100作为封闭通透液,室温放置30min。吸除封闭液,加入一抗(PECAM-1:内皮细胞标记物,Desmin:平滑肌细胞标记物),4℃孵育过夜。预冷的PBS洗3次。室温孵育二抗1h,预冷的PBS洗3次。滴加DAPI试剂染细胞核,室温避光5min,预冷的PBS洗3次。用含荧光淬灭剂的封片液进行封片,荧光显微镜下观察并分析MCECs纯度。
1.11CCK8法检测CYCSE对TM3细胞、MCECs细胞活力的影响
将TM3细胞以密度3×104/mL铺于96孔板中,分成6组:对照组、H2O2组、CYCSE组、ERK抑制剂(PD98059)+CYCSE组、AKT抑制剂(LY294002)+CYCSE组、西地那非组。两种抑制剂分别预处理1h后加入62.5μg/mL CYCSE处理24h,0.4mM H2O2处理2h。
将MCECs以密度1×104/mL铺于96孔板中,加入不同浓度CYCSE(31.3μg/mL和62.5μg/mL)、西地那非,处理24h,0.4mM H2O2处理2h。
两种细胞分别处理后加入CCK8,37℃下孵育1h。于酶标仪450nm处测量吸光度并计算细胞活力。
1.12TM3细胞中睾酮含量的测定
将TM3细胞以密度1×105/mL铺于6孔板中,62.5μg/mL CYCSE/西地那非预处理24h,0.4mM H2O2处理2h后,收集细胞培养基上清并离心去除沉淀。收集上清液,按小鼠ELISA检测试剂盒说明书检测TM3细胞中睾酮含量,用酶标仪在450nm处测吸光度值并计算。
1.13TM3细胞及MCECs中环磷酸鸟苷(cGMP)含量的测定
将TM3细胞以密度1×105/mL铺于6孔板中,62.5μg/mL CYCSE/西地那非预处理24h,0.4mM H2O2处理2h。将MCECs以密度5×104/mL铺于6孔板中,加入不同浓度CYCSE(31.3μg/mL和62.5μg/mL)处理24h,0.4mM H2O2处理2h。两种细胞分别处理完成后,收集细胞,用预冷的PBS洗涤2次,并加1mL PBS重悬细胞,在液氮中反复冻融6次,2500r离心20min并收集上清。按小鼠ELISA检测试剂盒说明书检测MCECs中cGMP含量,用酶标仪在450nm处测吸光度值并计算。
1.14TM3细胞中活性氧含量的测定
将TM3细胞以密度1×105/mL铺于6孔板中,62.5μg/mL CYCSE预处理24h,0.4mMH2O2处理2h后,收集细胞,用预冷的PBS洗涤两次,加DCFH-DA缓冲液混悬,37℃避光孵育20min。探针装载完成后,用预冷的PBS洗涤两次。每个样品加入300μL PBS混悬,利用流式细胞仪检测。
1.15TM3细胞中TGF-β1的表达
利用免疫荧光法检测TM3细胞中TGF-β1的表达。将TM3细胞以密度1×105/mL铺于6孔板中,62.5μg/mL CYCSE预处理24h,0.4mM H2O2处理2h后,吸除培养基,用预冷的PBS洗1次,加4%多聚甲醛室温固定15min,预冷的PBS洗1次。加含5%山羊血清的0.5%Triton X-100作为封闭通透液,室温放置30min。吸除封闭液,加入TGF-β1抗体,4℃孵育过夜。吸出一抗,室温孵育二抗1h,预冷的PBS洗2次。滴加DAPI试剂染细胞核,室温避光5min,预冷的PBS洗2次。荧光显微镜下观察并拍照。
1.16qRT-PCR
将TM3细胞以密度1×105/mL铺于6孔板中,62.5μg/mL CYCSE预处理24h,0.4mMH2O2处理2h后,将细胞收集到RNase free的ep管中,300g离心5min,弃去上清,加1mLTrizol,室温静置5min,12000r离心5min,弃沉淀;加200μL氯仿,振荡混匀,4℃条件下12000r离心15min;吸取上层水相加入等体积的异丙醇混匀,室温放置10min,4℃12000r离心10min,弃上清;加入300μL 75%冰乙醇,温和振荡,4℃8000r离心5min,弃上清;室温晾干;DEPC水溶解RNA沉淀,-20℃保存备用。制备琼脂糖凝胶,将Marker、样品与loadingbuffer混合物加入胶孔中,电泳结束后用凝胶成像仪观察,以确定RNA提取质量并测定RNA浓度。利用反转录试剂盒将RNA反转录成cDNA。GAPDH、Nrf2和NQO1的引物序列如表1。利用SYBR Green PCR Master Mix及PCR仪检测Nrf2、NQO1的转录水平。
表1 qRT-PCR的引物序列
1.17Western blotting
将TM3细胞或MCECs分别铺于6孔板中,分别加入CYCSE处理24h,0.4mM H2O2处理2h。收集细胞,PBS洗两次,弃上清。将液氮中的睾丸组织研磨成粉末。上述样品各加200μL RIPM裂解液(含1%PMSF),冰上裂解30min,4℃条件下12000r离心10min,取上清,-20℃备用。利用BCA蛋白含量检测试剂盒测定样本中蛋白含量,并分别调节蛋白浓度至一致。取提取的蛋白样品适量,加入等体积2X的上样缓冲液,混匀后煮沸10min,-20℃备用。各蛋白样品跑SDS-PAGE电泳并转印至NC膜上,于5%PBS脱脂奶粉中封闭1h,PBST洗膜3次,每次5min。除去PBST,加一抗溶液(GAPHD、Nrf2、HO-1、TGF-β1、SMAD2/3、ERK、p-ERK、AKT、p-AKT、eNOS、p-eNOS),4℃摇床孵育过夜。回收一抗,PBST洗膜3次,每次5min。除去PBST,加各蛋白对应种属的二抗溶液,摇床室温孵育1h。吸出二抗,PBST洗膜3次,每次5min。将ECL显色液A与显色液B等量混匀,均匀铺满NC膜,避光显色1min,于凝胶成像仪中显色、拍照并分析条带灰度值。
1.18数据统计与分析
所有实验重复三次,结果以平均值±标准差表示。用Graphpad Prism v6.0软件进行单向方差分析,p<0.05认为有统计学意义。
2.结果
2.1CYCSE的成分分析
CYCSE的组成成分包含36%蛋白质及62%淀粉。蛋白的分子量分布为32kDa、14.4kDa(图1,图2)。氨基酸自动分析仪检测CYCSE中氨基酸结果显示,CYCSE中含有17种氨基酸,包括人体所需8种氨基酸(表2)。
表2CYCSE中氨基酸含量
2.2CYCSE对肾阳虚大鼠勃起功能的改善及相关器官功能维护
利用氢化可的松(hydrocortisone,HCT)建立大鼠肾阳虚模型,于体内系统中明确了CYCSE对肾阳虚大鼠ED具有潜在治疗作用,且与器官功能的改善密切相关。
2.2.1对常规指标的影响
动物脏器重量的改变是重要的生物学特性指标之一,能够从一定程度上说明其功能的强弱。研究结果显示,模型组大鼠与正常组大鼠相比活动量减少、畏寒、扎堆、毛色无光泽、反应迟钝,并且体重、睾丸重量、附睾重量显著降低。CYCSE干预后,大鼠活动正常、毛色光泽恢复,体重、睾丸及附睾重量与模型组相比显著增加,且呈剂量依赖(表3)。
表3大鼠体重和器官重量
###P<0.001vs.对照组;*P<0.05,**P<0.01,***P<0.001vs.模型组
2.2.2对肾阳虚大鼠勃起功能的影响
山药蛋白提取物(CYCSE)对氢化可的松诱导的大鼠肾阳虚模型勃起功能障碍具有治疗作用,经实验结果表明是通过对海绵体组织形态的改善以及对海绵体平滑肌内皮细胞功能的修复和勃起关键信号途径(NO/cGMP)的激活来实现的。
2.2.2.1改善阴茎海绵体组织形态
海绵体在阴茎勃起过程中起着决定性的作用,其组织形态研究结果显示,与对照组相比,模型组海绵体平滑肌层薄、海绵窦紊乱且不连续,提示海绵体生理功能改变,不能正常行使勃起功能。而CYCSE干预后,可明显改善海绵体组织中平滑肌及内皮细胞排列不连续,间质细胞紊乱等现象(图3-图7)。
2.2.2.2修复阴茎海绵体平滑肌内皮细胞功能
阴茎海绵体平滑肌内皮细胞是控制勃起的关键细胞。本研究对模型的选择进行了优化。在以往研究中,通常选用人脐静脉内皮细胞(HUVECs),该细胞模型并不能准确模拟海绵体内皮细胞的微血管环境。而原代海绵体内皮细胞(MCECs),位于海绵体内表面,是维持海绵体功能最重要的细胞之一,是研究ED内皮功能的最佳选择。在细胞分离方式的选择上,多数选择酶分离法,但操作繁琐,纯度低,对细胞损伤力强,重复性差。Matrigel 3D培养系统,是一种新颖的非酶分离方法,能模拟体内细胞生长的三维环境,使MCECs与生长因子直接接触而诱导其从组织中爬出,保证MCECs原有形态及功能特性。而且操作省时、分离纯度高、重复性好,是研究ED内皮功能的最佳分离方案。图8为基于Matrigel 3D培养系统的MCECs的分离培养方案。
基于以上,采用Matrigel 3D培养系统分离小鼠原代阴茎海绵体内皮细胞MCECs,研究CYCSE对H2O2损伤MCECs的细胞活力的影响,采用CCK8法对细胞活力进行考察,由图9可知,CYCSE显著增加氧化损伤MCECs细胞的细胞活力,且呈剂量依赖。西地那非无挽救作用,于体外模型中进一步确证了CYCSE对ED具有改善作用。
2.2.2.3激活勃起关键信号途径(NO/cGMP)
为了进一步确定CYCSE与肾阳虚大鼠勃起功能之间的生理相关性,我们对控制勃起功能关键信号途径(NO/cGMP信号途径)进行了分析。非肾上腺素非胆碱能(NANC)机制是调节阴茎海绵体血管平滑肌舒张的主要机制,其中NO被认为是其主要的神经递质,NO/cGMP通路在阴茎勃起过程中起重要的调控作用。NANC神经末梢、血管内皮细胞和阴茎海绵体内皮细胞在一氧化氮合酶(NOS)的催化下释放一氧化氮(NO),通过细胞膜快速扩散入平滑肌细胞内,激活鸟苷酸环化酶,使环磷酸鸟苷(cGMP)合成增加,进而通过一系列级联反应诱发阴茎勃起。因此,NOS及cGMP是NO/cGMP信号途径的核心组成,其含量可用于阴茎勃起功能的评价。
在体内研究结果中,模型组阴茎海绵体中诱导型一氧化氮合酶(iNOS)和cGMP含量显著降低,而CYCSE干预后iNOS和cGMP两种生物标志物的含量显著增加,高剂量组与西地那非组无显著性差异(图10,图11)。
在海绵体内皮细胞中,内皮型一氧化氮合酶(eNOS)在钙离子的作用下被激活,进而调控NO/cGMP通路,促使阴茎勃起。然而在阴茎勃起的过程中,eNOS的钙依赖性非常短暂。AKT通路能直接引起eNOS磷酸化,降低对钙离子的需求,并进一步促使NO的产生,发挥器官功能。在体外研究结果中,CYCSE能促进氧化损伤MCECs中磷酸化的AKT及eNOS表达(图12-14),增加cGMP含量(图15),促使AKT/eNOS/cGMP级联反应发生,增强勃起功能。
2.2.3对肾阳虚大鼠睾丸功能的影响
山药蛋白提取物(CYCSE)改善肾阳虚大鼠睾丸功能的作用是通过改善睾丸形态、减少睾丸功能细胞凋亡、增加睾丸间质细胞含量、促进睾酮分泌、增强精子活力及改善睾丸纤维化六个方面共同确定的。
2.2.3.1改善睾丸组织形态
睾丸功能损伤是诱发ED的核心因素。因此为了探讨CYCSE对肾阳虚大鼠ED的改善作用是否与挽救睾丸功能相关,我们首先对睾丸形态进行考察。
利用HE染色法对大鼠睾丸进行形态学观察。模型组与对照组相比,睾丸曲细精管萎缩,生殖细胞层减少,模型组大鼠睾丸组织发生损伤。CYCSE能有效改善模型大鼠睾丸中曲细精管萎缩,增加生殖细胞层数,并呈剂量依赖(图16-20,表4)。
表4睾丸健康参数
##P<0.01vs.对照组;*P<0.05,**P<0.01vs.模型组
2.2.3.2减少睾丸功能细胞凋亡
细胞的发生与成熟过程和细胞凋亡密切相关,一定范围内的凋亡对机体具有积极的生理意义,但过度凋亡会使睾酮分泌明显减少,导致生精细胞凋亡增加,甚至不育。利用TUNEL法表征睾丸组织中功能细胞的凋亡情况,采用ImagePro软件对图像进行分析。由图21可知,模型组与对照组相比,肾阳虚大鼠睾丸组织中凋亡细胞数量增加。CYCSE干预后,两个浓度组的凋亡细胞数相比模型组减少约2-3倍。
活性氧自由基(reactive oxygen species,ROS)的过量产生与细胞的过度凋亡密切相关,是诱发细胞凋亡的核心因素。在多种内源或外源性的刺激下,ROS的生成或者清除速度受到破坏,导致过量ROS聚集破坏体内氧化还原平衡,引发氧化应激。氢化可的松可属于糖皮质激素,可刺激产生氧化应激,进而诱导细胞过度凋亡。因此,本部分研究进一步利用ROS,超氧化物歧化酶(SOD)和8-羟基-2-脱氧鸟苷(8-OHdG),评价CYCSE对肾阳虚大鼠睾丸组织及过氧化氢(H2O2)诱导氧化损伤的睾丸间质TM3细胞抗氧化应激的修复能力。结果可见,在模型组大鼠中,8-OHdG含量和SOD水平均偏离正常水平,表示睾丸处于氧化应激状态。CYCSE干预后,8-OHdG含量显著降低、SOD水平显著提高(图22-23)。利用流式细胞仪检测细胞ROS水平,由图24可知,CYCSE能够显著降低由于氧化损伤导致的TM3细胞内ROS过量释放。
Nrf2是调控细胞氧化应激反应的重要转录因子,同时也是维持细胞内氧化还原稳态的中枢调节者。Nrf2通过调控一系列抗氧化因子(如HO-1,NQO1)的表达,减轻活性氧和亲电体引起的细胞损伤,使细胞处于稳定状态,维持机体氧化还原动态平衡。本研究利用Western blot检测睾丸组织中Nrf2蛋白的表达情况,可见在模型组中,Nrf2蛋白的表达量显著高于对照组。CYCSE的保护能进一步增加损伤睾丸组织中Nrf2的表达,进而激活抗氧化防御系统(图25-26)。对TM3细胞中氧化应激关键调控靶点Nrf2及下游抗氧化应激因子的蛋白与转录水平进行检测分析,结果表明CYCSE可显著提高Nrf2转录及蛋白水平表达,并促进Nrf2向细胞核转移从而增加其下游因子HO-1及NQO1的表达,激活细胞抗氧化防御系统(图27-29)。
2.2.3.3增加睾丸间质细胞含量
睾丸间质细胞是内分泌性腺上皮细胞,是雄性动物体内产生睾酮最主要的细胞,是睾丸组织中最重要的功能细胞之一。在免疫组化实验中,利用3β-HSD特异性标记睾丸间质细胞,可见与对照组相比,模型组睾丸组织中3β-HSD免疫阳性细胞明显减少。不同浓度CYCSE干预后,睾丸间质细胞的免疫染色强度明显强于模型组,且高浓度组的作用效果强于西地那非(表5)。
表5睾丸间质细胞的半定量分析
Δ免疫染色强度采用简化量表评分,范围从阴性(-)到弱阳性(+)到强阳性(+++)。
进一步采用CCK8法考察体外睾丸间质TM3细胞活力,可见CYCSE能够显著增加氧化损伤TM3细胞的细胞活力,促进细胞增殖,且作用效果明显强于西地那非(图30)。已有研究表明,ERK及AKT信号途径是调控细胞增殖的核心控制途径。为了探知CYCSE促进TM3细胞增殖的作用是否与ERK和AKT信号途径相关,本研究引入了ERK抑制剂(PD98059)和AKT抑制剂(LY294002)。图30显示,两种抑制剂的加入显著阻滞了CYCSE对TM3细胞的保护作用。同时,Western blot分析结果显示,氧化损伤TM3细胞经CYCSE保护后p-ERK/ERK和p-AKT/AKT的表达量显著增加,而这种增加在两种抑制剂的调控下显著下调。说明CYCSE对于氧化损伤TM3细胞活力的挽救作用是通过激活ERK和AKT信号通路实现的(图31-33)。另外在氧化损伤的TM3细胞中发现,CYCSE的保护能显著增加由于H2O2的诱导的cGMP含量降低(图34)。
2.2.3.4促进睾酮分泌
睾酮是一种非常重要的雄性激素,主要由睾丸间质细胞分泌,是评价器官功能的重要指标。CYCSE能显著增加肾阳虚大鼠血清中睾酮含量,与睾丸组织中间质细胞含量显著增加结果相一致,且高剂量组的作用效果明显强于西地那非(图35)。
进一步检测TM3细胞中睾酮分泌量。由图36可知,由于H2O2的诱导作用,TM3细胞分泌睾酮的能力降低。CYCSE的保护能显著增加睾酮分泌量,且其作用效果明显强于西地那非。
2.2.3.5增强精子活力
精子活力是评价睾丸功能另一重要指标。由表6可知,模型组的精子数量明显减少,精子活力a级及a+b级的百分比与对照组相比减少约一半。CYCSE治疗后,精子数量,精子活力a级、a+b级百分比均明显增加,呈剂量依赖。且CYCSE高浓度组的三个指标均明显高于西地那非组。
表6精子数量及精子活力
##P<0.01vs.对照组;*P<0.05,**P<0.01,***P<0.001vs.模型组;ΔP<0.05,ΔΔP<0.01vs.CYCSE(80mg/kg)
2.2.3.6改善睾丸纤维化
睾丸纤维化是紊乱生精环境,破坏精子发生的重要原因,是导致睾丸功能损伤的关键。首先利用Masson染色分析睾丸组织中胶原纤维的表达(表7)。在模型大鼠中,大量胶原渗漏到间质组织中,睾丸组织发生纤维化。经CYCSE治疗后,随着给药浓度的增加胶原纤维显著减少。
表7睾丸纤维化的半定量分析
Δ免疫染色强度采用简化量表评分,范围从阴性(-)到弱阳性(+)到强阳性(+++)。
TGF-β1在组织修复和纤维化过程中起到信号刺激作用。TGF-β1通过启动细胞内信号级联传导,活化SMAD2/3形成复合体,进入细胞核内,调控胶原过度增殖而导致纤维化的发生。Western blot结果表明,CYCSE能够通过抑制TGF-β1/SMAD2/3信号通路,降低肾阳虚大鼠睾丸组织的纤维化程度(图37-38)。
利用免疫荧光法特异性标记TM3细胞中TGF-β1并分析表达量。与对照组相比,损伤TM3细胞中TGF-β1荧光强度明显增强,CYCSE保护后TGF-β1的过量表达被显著抑制(图39)。Western blot结果进一步表明,CYCSE能下调氧化损伤TM3细胞中TGF-β1/SMAD2/3信号通路的表达量,从而降低损伤TM3细胞的纤维化程度(图40-41)。
Claims (7)
1.一种山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用。
2.如权利要求1所述的山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用,其特征在于:所述勃起功能障碍为肾阳虚导致的勃起功能障碍。
3.如权利要求1所述的山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用,其特征在于:包含利用其为原料制备的具有改善和、或治疗勃起功能障碍作用的药品及健康产品,特别但不限于应用于改善和、或治疗哺乳动物勃起功能障碍。
4.如权利要求2所述的山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用,其特征在于:肾阳虚模型是基于氢化可的松诱导的大鼠肾阳虚模型,从对海绵体组织形态的改善以及对海绵体平滑肌内皮细胞功能的修复和勃起关键信号途径(NO/cGMP)的激活,证明山药蛋白提取物对肾阳虚大鼠勃起功能障碍(erectiledysfunction,ED)治疗的有效性。
5.如权利要求4所述的山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用,其特征在于:山药蛋白提取物改善肾阳虚大鼠睾丸功能的作用是通过改善睾丸组织形态、减少睾丸功能细胞凋亡、增加睾丸间质细胞含量、促进睾酮分泌、增强精子活力及改善睾丸纤维化六个方面共同确定的。
6.如权利要求1所述的山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用,其特征在于:所述山药蛋白提取物通过以下方式制备:取鲜山药用8-20倍量蒸馏水匀浆,静置1~4h,4~20℃,过滤,上清液调节pH至1~2,过滤,取沉淀,沉淀调节pH=7~8,冻干。
7.如权利要求6所述的山药蛋白提取物在制备治疗勃起功能障碍的药物中的应用,其特征在于:所述山药蛋白提取物通过以下方式制备:取鲜山药用15倍量蒸馏水匀浆,静置2h,4℃,过滤,上清液用HCl调节pH=2.0,过滤,取沉淀,沉淀用NaOH调节pH=7.0,冻干。
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