CN108310026A - 月见草油在改善多囊卵巢综合征慢性炎症方面的应用 - Google Patents
月见草油在改善多囊卵巢综合征慢性炎症方面的应用 Download PDFInfo
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Abstract
本发明公开了月见草油在改善多囊卵巢综合征慢性炎症方面的应用,是利用月见草油的特性和多囊卵巢综合征的病理特征,采用大鼠灌胃的方法,针对利用来曲唑构建的PCOS模型为研究对象,采用不同剂量的月见草油给予模型大鼠,改善模型大鼠炎症特征和炎症信号特征。因此通过本发明月见草油在改善多囊卵巢综合征的血清炎症水平和卵巢组织炎症表达方面研究的方法,将月见草油胶囊用于治疗多囊卵巢综合征疾病,不仅在患者生理上改善病情,并且在心理上给患者更多的快乐,同时患者复发率得到了降低,从而能够提高受孕率。
Description
技术领域:
本发明属于生物医药技术领域,特别涉及月见草油在改善多囊卵巢综合征慢性炎症方面的应用。
背景技术:
多囊卵巢综合征(PCOS)是孕龄妇女不排卵的常见原因,临床主要表现为月经失调、不孕、多毛与痤疮、肥胖、高血压、高脂血症以及糖代谢异常。研究证明:PCOS患者存在亚临床慢性炎症因子水平升高,PCOS的发病机制与慢性低度炎症性疾病有关。目前,治疗PCOS常用的西医方法为抗雄激素和增加胰岛素的敏感性,但临床上难以彻底治愈。中药能够明显调节内分泌紊乱,并在调经助孕方面有肯定的疗效,不良反应少,对西药促排卵药物抵抗的患者不失为一种有效的选择。
月见草油(Evening Primrese oil,EPO)是柳叶科植物月见草的种子油,是一种天然药物,因其含有70%人体正常生长所必需但自身又不能合成的不饱和脂肪酸,如大量γ-亚麻酸等,则在生殖、心血管、呼吸等各个领域都有作用。研究表明:EPO除了具有显著的抗氧化、降血脂、抗炎、减少血栓生成、降糖等作用,还具有防治骨质疏松症的作用。如CN200510078263.5公开的一种月见草油在防治骨质疏松症的药物制剂中的应用记载:发现几种植物油月见草油、紫苏籽油、麦胚芽油、核桃油、红花油具有防治骨质疏松症的作用,同时,又具有预防皮肤衰老的作用,可用于制备防治骨质疏松症的药物制剂,也可用于制备预防皮肤衰老的制剂。然而,目前未见有关于月见草油在改善多囊卵巢综合征炎症方面的应用。
发明内容:
本发明的目的旨在提供一种月见草油在改善多囊卵巢综合征的血清炎症水平和卵巢组织炎症表达方面研究的方法。
为达到上述目的,本发明采取以下技术方案:
月见草油在改善多囊卵巢综合征的血清炎症水平和卵巢组织炎症表达方面研究的方法,包括PCOS动物模型构建、PCOS动物模型鉴定、药物使用、药物治疗效果评价等,具体如下:
(1)分组及给药:
所有大鼠适应性喂养7天后,正常对照组大鼠用1%羧甲基纤维素灌胃,连续灌胃21天;模型组用来曲唑(1mg/kg+1%羧甲基纤维素钠)连续灌胃21天,在这期间所有老鼠均给予高脂饲料喂养;大鼠造模成功后,阳性对照组连续灌胃二甲双胍(60mg/kg)6周,治疗组按照大、中、小剂量每天灌胃月见草油胶囊连续6周,所有治疗组灌胃期间均给予正常饲料和饮用水。
按人和动物间体表面积折算的等效剂量比值表;二甲双胍治疗PCOS常用剂量为每次口服500mg,每日3次;换算后大鼠给药量为135mg/kg/d,连续用药4周。
(2)动情周期的观察:
大鼠的发情周期为4~5d,不同阶段阴道涂片脱落细胞呈现的细胞类型不同。吸取少量生理盐水,滴入大鼠阴道,吹打数次后吸出阴道液,再吹打均匀后进行涂片,在显微镜下观察记录细胞类型,并判断发情周期的阶段。
(3)大鼠体重和脂肪湿重的检测:
用电子体重秤称量大鼠体重和电子精密天平测量脂肪,计算脂肪湿重(脂肪湿重=卵巢周围脂肪+肾周围脂肪+肠系膜周围脂肪)。
(4)检测血清中胆固醇和三酰甘油水平:
各组大鼠禁食12h后称重,采尾静脉血用全自动生化分析仪测定血清胆固醇和三酰甘油水平。药物干预结束后再次测定以上指标。
(5)检测血清中炎症因子水平:
各组大鼠及药物干预最后一次给药后,实验动物禁食不禁水,次日经麻醉后进行心脏取血,全血样置4℃冰箱次日经3000g离心20min后收集上层血清并分装,依据ELISA试剂盒说明书通过酶标仪测定炎症因子浓度,剩余血清可放置至-80℃保存。
(6)HE染色鉴定PCOS模型卵巢组织形态改变:
各组大鼠灌药结束后禁食不禁水,第二天经水合氯醛(7%)麻醉后取其背部两侧卵巢,剥掉其表面的脂肪组织,多聚甲醛固定过夜,常规脱水,石蜡包埋,烤片结束后进行常规HE染色鉴定。
(7)免疫组化技术检测卵巢组织炎症信号蛋白表达定位:
将卵巢组织进行常规脱水,并切片后,60℃烤箱中烤片,经脱蜡液,复水,枸橼酸抗原修复,山羊血清封闭,炎症信号蛋白一抗4℃过夜孵育,DAB显色,苏木素复染,中性树脂封片晾干。
(8)免疫印迹技术检测炎症信号蛋白表达水平:
按照蛋白提取试剂盒提取各组大鼠的卵巢组织总蛋白,BCA测定蛋白定量,SDS-PAGE凝胶电泳,转膜并封闭,炎症信号蛋白一抗孵育,二抗孵育,ECL显影,并扫描拍照,图像分析。
以月见草油胶囊中月见草油不同剂量(试验中按照小剂量、中剂量和大剂量;灌胃剂量以60kg患者口服6粒,每天3次的月见草油胶囊换算)灌胃大鼠,以中剂量0.1g~1g为最佳剂量使用。
利用月见草油的特性和多囊卵巢综合征的病理特征,通过上述具体步骤采用大鼠灌胃的方法,针对利用来曲唑构建的PCOS模型为研究对象,采用不同剂量的月见草油给予模型大鼠,改善模型大鼠炎症特征和炎症信号特征,结果显示:
(1)按照中剂量PCOS模型大鼠灌胃,可改善PCOS模型大鼠体重和脂肪湿重;
(2)按照中剂量PCOS模型大鼠灌胃,可改善PCOS模型大鼠血清胆固醇和三酰甘油水平;
(3)按照中剂量PCOS模型大鼠灌胃,可改善PCOS模型大鼠血清炎症因子水平。
(4)按照中剂量PCOS模型大鼠灌胃,可改善PCOS模型大鼠卵巢组织炎症因子表达水平。
因此,通过本发明月见草油在改善多囊卵巢综合征的血清炎症水平和卵巢组织炎症表达方面研究的方法,将月见草油胶囊用于治疗多囊卵巢综合征疾病,不仅在患者生理上改善病情,并且在心理上给患者更多的快乐,同时患者复发率得到了降低,从而能够提高受孕率。
附图说明:
图1是实施例3中卵巢组织的HE染色特征;
图2是实施例4中IL-1β在各组卵巢组织中的表达定位;
图3是实施例4中NLRP3在各组卵巢组织中的表达定位;
图4是实施例5中westernblot检测NLRP3、Caspase-1和TNF-a的表达水平。
具体实施方式:
实施例1
PCOS模型大鼠血糖测定之后,大鼠禁食,于次日清晨称重,眼眶静脉采血,采集的标本血液静置30min后,2500r/min离心15min分离血清,将血清移入干净试管中,56℃水浴30min灭活,分装于1.5mL EP管,每管1mL,-20℃保存待测,用于ELISA检测血清IL-18、IL-10;将动物断颈椎脱曰,剖腹,取下肝脏、双侧卵巢及其周围脂肪组织。称取双侧卵巢质量及其周围脂肪组织湿重。将左侧卵巢组织迅速置于冻存管中,标记后液氮中保存,24h后置于-80℃保存。右侧卵巢组织置于甲醛溶液中固定,用于卵巢组织形态学观察。如表1、表2所示:
表1各组大鼠体重和脂肪湿重的比较
#表示与正常对照组差异显著p<0.05;*表示与模型组差异显著p<0.05
表2各组大鼠IL-18和IL-10水平变化的比较
#表示与正常对照组差异显著p<0.05;*表示与模型组差异显著p<0.05
结果显示:PCOS组血清IL-18水平明显高于正常组,IL-10水平却低于正常组,差异具有显著性;二甲双胍和中剂量月见草胶囊干预后血清IL-18水平与PCOS组相比明显下调,IL-10血清与PCOS组相比有所上调,上述炎症因子的变化可能参与PCOS的发病。
实施例2
卵巢组织甘油三酯(Triglyceride,TG)含量测定取50mg卵巢组织加入1mLPBS(Ig:20mL)匀浆,匀浆后加冰氯仿/甲醇(体积比2∶1)混合液2mL,旋涡振荡器充分混匀2min,在通风橱中风干过夜,再溶解于含60%丁醇和40%Triton-X114/甲醇(2∶1)的混合液中,溶解后用酶法(SerumTriglycerideDeterminationKit,TR0100,Sigma,USA)复孔测定TG含量。试验结束后采用日本奥林巴斯640型全自动生化分析仪检测各组大鼠血清总胆固醇(CHOL)、甘油三酯(TG),单位以每g卵巢组织中的TG含量(mg/g)来表示。如表3所示:
表3各组大鼠胆固醇和三酰甘油的比较
#表示与正常对照组差异显著p<0.05;*表示与模型组差异显著p<0.05
结果显示:二甲双胍干预有明显降低大鼠体质量的趋势,差异没有统计学意义。PCOS组大鼠血清CHOL和TG含量显著高于正常对照组(P<0.05)。二甲双胍和中剂量月见草胶囊干预后大鼠血清CHOL和TG含量显著降低,与PCOS组比较,差异有统计学意义(P<0.05)。
实施例3
灌胃处理完成后处死大鼠,将各组大鼠一侧卵巢组织行HE染色制片,观察卵巢组织形态学(参见图1)。
(1)正常对照组:大鼠卵巢皮髓质构造正常,可见较多的始基卵泡及各级发育中的卵泡和黄体,排卵前卵泡体积大,卵泡液含量多。
(2)模型空白对照组:肉眼观察大鼠卵巢体积较正常对照组明显增大,长径较长;镜下见大鼠卵巢组织内有较多数量的囊状扩张卵泡,卵泡内颗粒细胞层数显著减少甚至消失,不见卵母细胞及放射冠,黄体和发育各阶段的卵泡数量显著减少,卵泡膜细胞及间质细胞可见增生。
(3)二甲双胍组:肉眼观察大鼠卵巢体积较模型空白对照组稍缩小;镜下见大鼠卵巢原始卵泡及初级卵泡数量增多,黄体数量增加。
(4)月见草油组:肉眼观察大鼠卵巢体积也较模型空白对照组稍缩小;镜下见大鼠卵巢原始卵泡及初级卵泡数量增多,黄体数量增加。
实施例4
石蜡切片免疫组化染色按照试剂盒说明书操作:将卵巢组织冲洗干净,脱水、包埋、连续切片,片厚5μm;石蜡切片脱蜡至水;3%H2O2室温孵育10min,PBS冲洗5min×3次;滴加适当比例稀释的IL-1β、NLRP3抗体,37℃孵育2h,PBS冲洗5min×3次;25℃孵育2h,4℃过夜,PBS洗2min×3次;滴加生物素标记羊抗兔IgG,室温下20min,PBS洗2min×3次;滴加SABC复合物,室温20℃,PBS洗5min×4次。DAB显色10min左右,蒸馏水多次洗涤,苏木素轻度复染,脱水,透明,中性树胶封片,显微镜下观察。阴性对照不加一抗,以正常山羊血清和PBS代替IL-1β、NLRP3抗体作孵育。
参见图2、图3,结果显示:IL-1β、NLRP3蛋白在各组颗粒细胞、黄体细胞和间质细胞中均有表达,NLRP3在各个细胞的细胞浆中表达,IL-1β在细胞核和细胞浆中均有表达。
实施例5
将组织从-70℃冰箱中取出,并准备液氮及研磨工具,将研磨过的卵巢组织装入1.5mL离心管内并称重,加入增强型RIPA与PMSF混合液(两者比例100∶1),加入混合液的量与组织的比重为5∶1,放入4℃高速离心机下13000r/min离心30min,取上清,即为所提取之大鼠卵巢组织总蛋白。BCA法蛋白定量,调整使各样品总蛋白含量一致,采用5倍上样缓冲液蛋白变性,5%浓缩胶及10%分离胶SDS-PAGE凝胶电泳,PVDF膜转印,5%TBST脱脂奶粉封闭2小时,加入IL-1β、NLRP3抗体(工作浓度1∶400)4℃孵育过夜;洗涤后加入相应辣根过氧化酶标记的Ⅱ抗(工作浓度1∶10000),37℃孵育2h;ECL法显色,胶片压片。采用Photoshop图像分析软件分析蛋白条带灰度值,以β-actin作为内参照蛋白进行校准,采用目标蛋白条带灰度值β-actin来表示蛋白相对表达水平。
参见图4,结果显示:NLRP3、Caspase-1和TNF-a蛋白在各组卵巢组织中均有表达,其中PCOS模型组,机体呈现慢性炎症状态,NLRP3、Caspase-1和TNF-a表达较高,而当二甲双胍和月见草胶囊治疗后NLRP3、Caspase-1和TNF-a表达降低。
Claims (3)
1.月见草油在改善多囊卵巢综合征慢性炎症方面的应用。
2.根据权利要求1所述的月见草油在改善多囊卵巢综合征慢性炎症方面的应用,其特征在于:所述月见草油制备成月见草油胶囊,其剂量为0.1g~1g。
3.根据权利要求2所述的月见草油在改善多囊卵巢综合征慢性炎症方面的应用,其特征在于:所述月见草油胶囊与羧甲基纤维素钠制备成月见草油混悬液,用于改善多囊卵巢综合征的血清炎症水平和卵巢组织炎症表达。
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