CN117643592B - 人参皂苷20(R)-25-OH-Rg2促进卵巢修复的用途 - Google Patents
人参皂苷20(R)-25-OH-Rg2促进卵巢修复的用途 Download PDFInfo
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Abstract
本发明公开了人参皂苷20(R)‑25‑OH‑Rg2促进卵巢修复的用途。本发明通过实验发现人参皂苷20(R)‑25‑OH‑Rg2可通过激活Nrf 2/HO‑1信号通路来改善相关炎症因子水平，促进了POI大鼠卵巢功能的修复，有效改善患者卵巢功能，缓解患者不良情绪，可用于制备促进卵巢修复的药物和保健品。
Description
技术领域
本发明涉及人参技术领域，具体是涉及一种人参皂苷20(R)-25-OH-Rg2促进卵巢修复的用途。
背景技术
卵巢功能不全是指已建立规律月经的妇女，在40岁以前，因卵巢功能衰退而出现持续性闭经和生殖器官萎缩，常伴有促性腺激素水平上升和雌激素下降，临床表现为闭经、不孕，不同程度的潮热多汗、阴道干涩、情绪波动、头晕、失眠及性欲下降等卵巢功能低落的类更年期症状；其发病率为1-2％，发病原因尚未完全明确，90％的病例为特发性的，10％的与自身免疫、感染、基因和医源性等有关。卵巢功能不全远期危害大，因失去雌激素的保护作用而使罹患骨质疏松、冠心病的风险急剧上升。
目前卵巢功能不全尚无理想的治疗方法。
发明内容
本发明目的是克服现有技术的不足，提供一种人参皂苷20(R)-25-OH-Rg2促进卵巢修复的用途。为实现上述目的，本发明提供了以下技术方案：
第一方面，本发明提供了人参皂苷20(R)-25-OH-Rg2促进卵巢修复的用途。
第二方面，本发明还提供了人参皂苷20(R)-25-OH-Rg2用于制备促进卵巢修复的药物的用途。
第三方面，本发明还提供了人参皂苷20(R)-25-OH-Rg2用于制备促进卵巢修复的保健品的用途。
本发明与现有技术不同之处在于本发明取得了如下技术效果：
本发明通过实验发现人参皂苷20(R)-25-OH-Rg2可通过激活Nrf 2/HO-1信号通路来改善相关炎症因子水平，促进了POI大鼠卵巢功能的修复，有效改善患者卵巢功能，缓解患者不良情绪，可用于制备促进卵巢修复的药物和保健品。
附图说明
图1是本发明各组大鼠血清性激素含量对比；其中，A为血清E2，B为血清LH，C为血清FSH，@表示与对照组对比P＜0.05，#表示与模型组对比P＜0.05，￥表示与低剂量人参皂苷对比P＜0.05；&表示与高剂量人参皂苷对比P＜0.05；
图2是本发明各组大鼠炎症因子含量对比；其中，A为IL-4，B为IL-6，C为TNF-α，@表示与对照组对比P＜0.05；#表示与模型组对比P＜0.05；￥表示与低剂量人参皂苷组对比P＜0.05；&表示与高剂量人参皂苷组对比P＜0.05；
图3是本发明各组大鼠HE染色(100μm)；其中，A为对照组，B为模型组，C为低剂量人参皂苷组，D.高剂量人参皂苷组，E.西药组；
图4是本发明各组大鼠卵巢组织Nrf 2/HO-1蛋白表达水平对比；其中，A为Nrf 2，B为HO-1，@表示与对照组对比P＜0.05，#表示与模型组对比P＜0.05，￥表示与低剂量人参皂苷组对比P＜0.05，&表示与高剂量人参皂苷组对比P＜0.05；
图5是本发明卵巢组织Nrf 2/HO-1蛋白检测；其中，A为对照组，B为模型组，C为低剂量人参皂苷组，D为高剂量人参皂苷组，E为西药组；
图6是本发明患者焦虑、抑郁情绪评分对比；其中，A为SAS评分，B为SDS评分，*表示组间同时间比较P＜0.05。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用材料、试剂等，如无特别说明，均可从商业途径获得。
一、实验材料
取60只8周龄SPF级雌性SD大鼠(浙江省维通利华实验动物技术有限公司)，体质量(220±20)g。于SPF级饲养室适应7d，再连续进行7d阴道涂片判断动情周期，60只大鼠均有规律动情周期。饲养环境：温度为(22±2)℃，相对湿度为50％～70％，昼夜各12h。研究过程中对动物的处置符合动物委员会的准则。
基础实验所用试剂包括：雷公藤多苷片(浙江得恩德制药有限公司，国药准字Z33020422)；人参皂苷20(R)-25-OH-Rg2纯度不低于98％；戊酸雌二醇片(拜耳医药保健有限公司广州分公司)；E2、FSH、LH的ELISA试剂盒(美国Ominm Abs公司)；肿瘤坏死因子-α、白细胞介素-4和白细胞介素-6ELISA试剂盒(美国R&D公司)；兔抗β-actin多克隆抗体、兔抗Nrf 2和HO-1单克隆抗体，HRP-Linked Antibody均购自Cell Signaling Technology。基础实验所用仪器包括：BX51型光学显微镜(日本Olympus公司)；Bio-Rad 1658001型垂直电泳仪(美国Bio-Rad公司)。
二、实验方法
1、基础实验
取60只SD雌性大鼠，随机抽取12只作为空白对照组，其余48只通过雷公藤多苷片建立POI(卵巢功能不全)模型，造模成功后随机分为模型组、低、高剂量人参皂苷组及西药组，每组各12只。低、高剂量人参皂苷组分别给予25、100mg·kg-1人参皂苷20(R)-25-OH-Rg2，连续灌胃15d；西药组给予0.12mg·kg-1戌酸雌二醇，连续灌胃15d；对照组及模型组均给予等量生理盐水，灌胃15d。测定各组大鼠血清中E2、LH和FSH水平；采用HE染色观察各组大鼠卵巢组织的病理变化；采用Western blot检测各组大鼠卵巢组织中Nrf 2和HO-l蛋白表达情况。
2、临床实验
选择接受子宫肌瘤切除手术治疗的70例患者作为研究对象，根据患者接受手术时间的先后顺序将其均分为对照组与观察组，每组各35例。对照组患者均为女性，年龄42-57岁，平均年龄(49.37±3.68)岁；BMI 18.57-25.89kg/m2，平均(21.31±1.25)kg/m2；受教育程度：大专及以下18例、本科及以上12例；病程：1-6年，平均(4.22±0.36)年；肌瘤直径：5-10cm，平均(5.49±1.24)cm；住院时间：5-19d，平均(9.30±3.05)d。观察组患者均为女性，年龄42-57岁，平均年龄(48.40±3.15)岁；BMI 18.39-25.77kg/m2，平均(22.49±1.32)kg/m2；受教育程度：大专及以下19例、本科及以上11例；病程：1-7年，平均(4.51±0.47)年；肌瘤直径：5-10cm，平均(5.28±1.15)cm；住院时间：6-11d，平均(8.13±1.36)d。给予对照组患者加速康复外科护理模式干预，给予观察组患者人参皂苷20(R)-25-OH-Rg2药剂联合加速康复外科护理模式干预。获得医院伦理委员会批准，所有患者均已签署知情同意书。
3、研究对象入组标准
纳入：1)患者均符合子宫肌瘤诊断标准，并经彩超、核磁共振等检查确认；2)患者的精神状态正常，意识清醒，具备正常沟通交流学习的能力；3)患者年龄均≥18岁。
排除：1)合并其它恶性肿瘤、免疫系统、血液系统、脏器功能等疾病；2)存在精神类疾病或行为、认知功能障碍性疾病；3)接受过其它子宫肌瘤药物治疗；4)因自身原因，无法完整配合研究进行。
三、大鼠实验
1、大鼠POI模型建立
随机选取48只SD雌性大鼠给予配制好的雷公藤多苷片(40mg·kg-1·d-1)悬浮液灌胃，连续给药30d进行造模。每天上午8:00给药，下午4:00进行阴道细胞涂片，观察、记录动情周期，动情周期平均为90～120h，不在此范围的则为动情周期紊乱，当大鼠动情周期全部紊乱，表明建模成功。
2、分组与给药
将48只建模成功的SD大鼠随机分为模型组、低、高剂量人参皂苷组及西药组，每组各12只。低、高剂量人参皂苷组分别给予25、100mg·kg-1人参皂苷20(R)-25-OH-Rg2悬浮液(生理盐水配制)灌胃；西药组给予0.12mg·kg-1戌酸雌二醇灌胃；对照组及模型组均给予10mg·kg-1生理盐水灌胃。各组给药每天1次，连续3周。
3、指标检测
3.1、样本采集
给药3周后，将大鼠麻醉，开腹，腹主动脉取血。静置4h，以3000r·min-1离心15min，分离血清，-80℃保存。采血完毕，处死大鼠，分离出双侧卵巢组织，一侧置于质量浓度为4g·mL-1的多聚甲醛液中固定24h备用，另一侧置于-80℃保存备用。
3.2、血清性激素测定
ELISA按照E2、FSH、LH试剂盒说明书操作，加入对照品工作液100μL，加入待测样品100μL至其他孔，覆膜孵育90min。弃去液体甩干，加入生物素化抗体工作液100μL，覆膜孵育60min。加人洗涤液350μL，重复洗板3次。加入酶结合物工作液100μL，覆膜温育30min，洗板5次。加入底物溶液90μL，覆膜避光孵育15min，加入终止液50μL。用酶标仪在450nm波长处测量各孔的吸光度值(A)。建立标准曲线，计算样本的质量浓度。
3.3、血清炎症因子测定
ELISA按照II-4、II-6和TNF-α试剂盒说明书操作，取EP管加入对照品稀释液100μL，制备6个梯度质量浓度对照品。样品孔中加入样本40μL，再加工作液10μL。各孔依次加酶标试剂50μL(空白孔除外)，密封后置于37℃水浴锅中孵育30min。加入洗涤液，洗涤5次并拍干。加显色剂A和B50μL，避光显色10min，终止反应。用酶标仪在波长450nm处依序测量各孔吸光度值。建立标准曲线，计算样本的质量浓度。
3.4、HE染色
将用质量浓度为4g·mL-1的甲醛固定24h的卵巢组织用梯度体积分数乙醇脱水、石蜡包埋、切片、脱蜡，苏木素染色5～8min，清水冲洗1～2min，盐酸酒精分化1～5s，伊红染色3～5min，清水冲洗1～2min，梯度体积分数乙醇脱水，蒸馏水冲洗，脱水，二甲苯透明，中性树胶封片。置于阴凉处晾干，将组织切片置于光学显微镜下观察卵巢组织形态变化。
3.5、Nrf 2/HO-1蛋白表达量检测
取180mg卵巢组织加人适量RIPA裂解液提取蛋白，用BCA蛋白定量试剂盒检测蛋白质量浓度，蛋白变性后进行凝胶电泳分离，80V电转1.5h，转至PVDF膜上，质量浓度50g·L-1脱脂牛奶封闭2h(室温)，分别加人1:1 000稀释Nrf 2、HO-1和β-actin抗体，置于摇床孵育过夜(4℃)，次日用TBST缓冲液洗膜10min×3次，加人1:2000稀释HRP标记二抗，摇床孵育2h(室温),再次用TBST缓冲液洗膜10min×3次，于暗室中滴加ECL发光试剂孵育1～2min,凝胶分析系统曝光分析。用Image J软件分析条带灰度值，以目的蛋白Nrf 2、HO-1与内参β-actin灰度值的比值作为蛋白的相对表达量。
四、临床实验
1、临床指标
(1)焦虑、抑郁情绪：于患者入院时及出院1周、2周、1个月后，采用焦虑自评量表(Self-Rating Anxiety Scale，SAS)及抑郁自评量表(Self-Rating Depression Scale，SDS)对其负面情绪进行评估，分值与情绪状态成反比，即得分越低情绪状态越好。
(2)卵巢功能：于手术前、手术后(出院一个月)，各抽取患者清晨空腹肘静脉血5mL，采用ADVIACentaurXP全自动化学发光免疫分析仪(美国)及其配套试剂进行雌二醇(E2)、黄体生成素(LH)和卵泡刺激素(FSH)水平检测。
2、统计学方法
制图软件选用GraphPad Prism 8；采用SPSS25.0对数据进行分析；计量资料运用t检验对比，表示；计数资料运用x2检验对比，n(％)表示。以P＜0.05表示对比有统计学意义。
五、实验结果
1、各组大鼠血清性激素含量对比
如图1所示，与对照组相比，模型组的E2表达水平降低，LH、FSH表达水平增加(P＜0.05)；与模型组相比，其余3组的血清E2表达水平增加，LH、FSH表达水平降低(P＜0.05)；与低剂量人参皂苷组相比，高剂量人参皂苷组和西药组的血清E2表达水平增加，LH、FSH表达水平降低(P＜0.05)；人参皂苷组与西药组上述指标相比，差异无统计学意义(P＞0.05)。
2、各组大鼠炎症因子含量对比
如图2所示，与对照组相比，模型组的IL-4、IL-6、TNF-α表达水平增加(P＜0.05)；与模型组相比，其余3组的IL-4、IL-6、TNF-α表达水平降低(P＜0.05)；与低剂量人参皂苷组相比，高剂量人参皂苷组和西药组的IL-4、IL-6、TNF-α表达水平降低(P＜0.05)；高剂量人参皂苷组与西药组上述指标相比，差异无统计学意义(P＞0.05)。
3、各组大鼠HE染色情况
如图3所示，对照组大鼠卵巢结构清晰，各级卵泡数量较多，发育正常；模型组大鼠卵巢组织萎缩明显，各级卵泡数量减少，卵泡结构破坏严重；与模型组比较，低、高剂量人参皂苷组和西药组卵巢组织发育情况有所改善，可见各级生长卵泡数量增加，闭锁卵泡数量减少。与低剂量人参皂苷组比较，高剂量人参皂苷组和西药组原始卵泡和窦状卵泡的数量显著增加，闭锁卵泡的数量减少。
4、各组大鼠卵巢组织Nrf 2/HO-1蛋白表达水平对比
如图4、5所示，与对照组相比，模型组的Nrf 2、HO-l蛋白表达水平降低(P＜0.05)；与模型组相比，其余3组的Nrf 2、HO-l蛋白表达水平增加(P＜0.05)；与低剂量人参皂苷组相比，高剂量人参皂苷组和西药组的Nrf 2、HO-l蛋白表达水平增加(P＜0.05)；高剂量人参皂苷组与西药组上述指标相比，差异无统计学意义(P＞0.05)。
5、患者焦虑、抑郁情绪评分对比
如图6所示，入院时，两组患者的SAS、SDS评分对比(P＞0.05)；出院1周、2周及1个月后的随访调查中，观察组的SAS、SDS评分水平均明显低于对照组(P＜0.05)。
6、患者卵巢功能情况对比
手术前，两组患者的血清E2、LH、FSH水平对比(P＞0.05)；手术后，观察组的血清E2水平明显低于对照组，LH、FSH水平则明显高于对照组(P＜0.05)，详见表1。
表1患者卵巢功能情况对比
六、结论
本发明使用雷公藤多苷片建立POI大鼠模型,结果显示,与对照组相比，模型组的E2表达水平降低，LH、FSH表达水平增加，出现了动情周期紊乱、卵巢形态受损等症状，与POI的症状一致。在给予人参皂苷20(R)-25-OH-Rg2与西药(戊酸雌二醇片)干预后，大鼠E2水平均明显增加而LH、FSH水平明显下降，卵巢形态受损情况也得到了改善，表明人参皂苷20(R)-25-OH-Rg2与戊酸雌二醇片均可在一定程度上提高POI大鼠的性激素水平。高剂量人参皂苷组和西药组血清中炎性因子IL-4、IIL-6和TNF-α水平均降低，而Nrf 2和HO-1的蛋白表达水平较模型组明显升高，可见激活Nrf 2/HO-1信号通路，改善炎性因子水平，对POI大鼠卵巢功能有积极影响，结合大鼠血清中性激素与卵巢组织形态变化，表明人参皂苷20(R)-25-OH-Rg2对POI大鼠卵巢的保护作用与Nrf2/HO-1信号通路介导的炎性调节有关。基于动物实验结果，人参皂苷20(R)-25-OH-Rg2对雌性卵巢功能具有很好的改善作用。针对患者手术后，观察组的血清E2水平明显低于对照组，LH、FSH水平则明显高于对照组(P＜0.05)，此结果与上述动物实验结果相似，提示了人参皂苷20(R)-25-OH-Rg2的使用可进一步改善子宫肌瘤切除术患者的卵巢功能。此外，在患者术后焦虑、抑郁情绪方面，本研究结果显示，出院1周、2周及1个月后的随访调查中，观察组的SAS、SDS评分水平均明显低于对照组(P＜0.05)，此结果表明了，相较于单纯加速康复外科护理，人参皂苷20(R)-25-OH-Rg2的联合使用可进一步缓解患者术后焦虑、抑郁等负面情绪，促进伤口的恢复。
因此，人参皂苷20(R)-25-OH-Rg2可通过激活Nrf 2/HO-1信号通路来改善相关炎症因子水平，促进了POI大鼠卵巢功能的修复，有效改善患者卵巢功能，缓解患者不良情绪。
以上所述的实施例仅仅是对本发明的优选实施方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案作出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
Claims (1)
1.人参皂苷20(R)-25-OH-Rg2作为唯一的活性成分用于制备促进卵巢功能不全的卵巢修复的药物的用途。
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