CN116251090A - 一种木犀草素缓解口干症状的方法 - Google Patents
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Abstract
本发明提供一种木犀草素缓解口干症状的方法,涉及生物工程领域。本发明包括如下步骤:第一步、以AQP5启动子为靶点进行双荧光素酶报告基因活性检测的药物筛选;第二步、测试木犀草素上调涎腺细胞AQP5转录水平;第三步、进行蛋白印迹和免疫荧光实验,测试木犀草素促进涎腺细胞AQP5蛋白表达水平;第四步、建立去卵巢小鼠动物模型模拟绝经期口干症,分组进行灌胃给药;第五步、对各组小鼠唾液分泌指标进行分析;第六步、验证缓解了口干症状;第七步、验证木犀草素对小鼠各组织无害。本发明通过口干症动物模型去卵巢鼠验证木犀草素促进唾液分泌,治疗绝经期口干症的作用。
Description
技术领域
本发明涉及一种绝经期口干症的治疗方法,具体为一种木犀草素缓解口干症状的方法,属于生物工程技术领域。
背景技术
正常人每日分泌1000-1500毫升的唾液;正常人平均静态唾液流速为0.3-0.4mL/min,刺激性唾液流速为1.5-2.0mL/min,当静态唾液流速≤0.1mL/min,刺激性唾液流速≤0.5-0.7mL/min时,可诊断为唾液腺功能减退,即口干症。
口干症是由于唾液分泌障碍导致的临床常见疾病,一般情况下,当唾液分泌的速率小于口腔黏膜吸收唾液的速率和唾液蒸发的速率之和时,就会感到口干,只要患者以口干为主诉就诊,就可诊断为口干症。口干症可导致口腔局部猖獗龋、牙周病及灼口综合征等,影响患者的语音功能、咀嚼功能、吞咽功能及义齿佩戴。口干症的患病率高,约为5.5%-46%;50%以上绝经期女性有自觉口干症状。口干症发病率高且绝经期女性高发,严重影响患者生活质量。然而,临床尚缺乏治疗的有效手段,研究治疗口干症的有效药物是重要的研究课题。
目前,根据唾液分泌功能障碍的程度,有不同的治疗方法来恢复唾液分泌功能,减轻症状,一般来说,这些途径可以分为内源性和外源性:内源性途径是通过药物或基因治疗替代或增强唾液腺功能;外源性方法包括局部应用唾液替代品,以替代自然唾液,但功能有限。在口干症的内源性治疗中,药物治疗主要包括胆碱能受体激动剂和激素治疗。胆碱能受体激动剂主要包括匹鲁卡品、西维美林等,可刺激胆碱能受体增加唾液分泌,用于缓解口干症状。然而,研究报道,匹鲁卡品等胆碱能受体激动剂可引起不良的心血管和肺效应,还会引起恶心和头晕。此外,针对绝经期口干症,雌激素治疗可显著刺激唾液分泌。M.L.Lago等进行了一项研究,涉及86名绝经后妇女,结果表明,雌激素治疗对唾液流量的增加有较大的影响。激素替代疗法可促进绝经后妇女的唾液流量。尽管雌激素可促进唾液分泌,缓解绝经期口干症状,然而其长期应用导致患子宫内膜癌及乳腺癌风险显著增高。
因此,深入研究唾液分泌的关键靶点寻找有效且副作用小的治疗口干症的药物是重要的研究课题。
发明内容
(一)解决的技术问题
本发明的目的就在于为了解决上述问题而提供一种木犀草素缓解口干症状的方法,以解决现有技术中的问题。
(二)技术方案
为实现以上目的,本发明通过以下技术方案予以实现:一种木犀草素缓解口干症状的方法,包括如下步骤:
第一步、以AQP5启动子为靶点进行双荧光素酶报告基因活性检测的药物筛选;
用293T细胞以105密度铺48孔板,将荧光素酶报告基因pGL3/AQP5p质粒转染细胞,用1μM的10种黄酮类天然产物作用细胞24小时,应用PBS作为阴性对照,应用0.1μM雌二醇作为阳性对照。双荧光素酶报告基因检测试剂盒进行检测,所得的结果为各个实验样品的荧光素酶的活性与Renilla荧光素酶活性的比值;
确认可以显著激活AQP5启动子的天然产物单体木犀草素;
第二步、测试木犀草素上调涎腺细胞AQP5转录水平;
用HSG细胞以106密度铺6孔板,用不同浓度木犀草素(0.01μM、0.1μM、1μM)作用细胞48小时,应用0.1μME2作为阳性对照,用TRlzol提取细胞内总RNA,合成cDNA,应用hsAQP5引物(hsAqp5上游引物5-TGCCATCCTTTACTTCTACCTG-3,下游引物5-CTCATACGTGCCTTTGATGATG-3)进行Realtime-PCR;
第三步、进行蛋白印迹和免疫荧光实验,测试木犀草素促进涎腺细胞AQP5蛋白表达水平;
用HSG细胞以106密度铺6孔板,用不同浓度木犀草素(0.01μM、0.1μM、1μM)作用细胞48小时,应用0.1μME2作为阳性对照,应用RIPA裂解细胞收蛋白样,westernblot检测在不同浓度木犀草素作用下AQP5蛋白的表达水平;
用HSG细胞以5×105密度铺12孔板,用1μM木犀草素作用细胞48小时,应用0.1μME2作为阳性对照。通过免疫荧光染色检测木犀草素对HSG细胞AQP5表达及定位的影响,用AQP5抗体进行免疫荧光染色(红色),细胞核用DAPI染色;
第四步、建立去卵巢小鼠动物模型模拟绝经期口干症,分组进行灌胃给药;
首先,建立去卵巢小鼠动物模型,模拟绝经期口干症,取雌性ICR小鼠共24只,将小鼠随机分为4组,每组6只,分为假手术/生理盐水组、去卵巢/生理盐水组、去卵巢/木犀草素组、去卵巢/雌二醇组;
其次,对6只小鼠进行假手术,对另外18只小鼠进行去卵巢手术。去卵巢手术小鼠麻醉后在腹部正中做切口,然后切除双侧卵巢,假手术暴露卵巢,移除一小块脂肪组织,切口分层缝合。手术两周后,将去卵巢小鼠进一步按照给药进行分组,小鼠每日接受灌胃给药。将假手术的6只小鼠给予生理盐水(假手术/生理盐水组),将去卵巢的18只小鼠分为3组(每组6只),分别给予生理盐水(去卵巢/生理盐水组)、木犀草素(去卵巢/木犀草素组)、雌二醇(去卵巢/雌二醇组),共给药12周;
最后,以唾液分泌量和饮水量检测评价小鼠口干症唾液腺功能指标;
第五步、对各组小鼠的唾液分泌指标进行分析;
第六步、验证木犀草素降低去卵巢小鼠的饮水量,缓解口干症状;
第七步、在给药12周后,处死小鼠,取小鼠心、肺、肝、肾组织进行组织学染色,验证木犀草素对小鼠各组织无害。
优选地,在给药前和给药后4周、8周、12周测量小鼠唾液分泌及饮水量,计算各组小鼠唾液分泌指数和周饮水指数的平均值,观察各组之间的变化。
优选地,根据结构比对,从天然产物库选择30种天然产物作为实验药物,包括黄酮类天然产物10种、滋阴养血生津中药单体10种、抗炎抗肿瘤天然产物单体10种;
首先,需要筛选对AQP5启动子具有激活作用的10种黄酮类天然产物;
其次,采用相同的实验方法筛选对AQP5启动子具有激活作用的10种滋阴养血生津中药单体;
最后,采用相同的实验方法筛选对AQP5启动子具有激活作用的10种抗炎抗肿瘤天然产物单体。
优选地,其中,实验结果均为3次独立的实验结果的平均值,每次实验每组设计3个平行孔,所有的结果均以平均值±标准差(mean±S.D.)表示。
优选地,木犀草素激活AQP5启动子是PBS对照组的约4.2倍,10种中药单体均可激活AQP5启动子,与PBS对照组比较均具有统计学差异;10种抗炎抗肿瘤天然产物单体对AQP5启动子激活作用不显著。
优选地,应用PBS作为阴性对照,应用0.1μM雌二醇及1μM木犀草素作为阳性对照。
本发明提供了一种木犀草素缓解口干症状的方法,其具备的有益效果如下:
1、本发明以唾液分泌调控的核心靶点水通道蛋白5(AQP5)为靶点,根据口干症的病因及疾病特点,选择30种天然产物作为实验药物,包括黄酮类天然产物10种、滋阴养血生津中药单体10种、抗炎抗肿瘤天然产物单体10种,筛选出显著活化AQP5启动子的天然产物木犀草素;并进一步研究其在细胞水平调控AQP5的作用机制;通过口干症动物模型去卵巢鼠验证木犀草素促进唾液分泌,治疗绝经期口干症的作用。
附图说明
图1为本发明的10种黄酮类天然产物激活AQP5启动子活性的示意图;
图2为本发明的10种滋阴养血生津中药单体激活AQP5启动子活性的示意图;
图3为本发明的10种抗炎抗肿瘤天然产物单体激活AQP5启动子活性的示意图;
图4为本发明的木犀草素在涎腺细胞对AQP5转录水平作用的对照图;
图5为本发明的AQP5蛋白表达水平对照图;
图6为本发明的木犀草素作用下HSG细胞AQP5染色强度对照示意图;
图7为本发明的对小鼠实验组分析对比图;
图8为本发明的不同组小鼠唾液分泌指数变化参照图;
图9为本发明的12周小鼠唾液分泌指数柱状图;
图10为本发明的小鼠饮水指数折线图;
图11为本发明的12周小鼠饮水指数柱状图;
图12为本发明的木犀草素化学式表示图;
图13为本发明的小鼠组织学染色对照图;
图14为本发明的整体设计思路示意图。
具体实施方式
本发明实施例提供一种木犀草素缓解口干症状的方法。
包括以下步骤:第一步、以AQP5启动子为靶点进行双荧光素酶报告基因活性检测的药物筛选。
根据结构比对,从天然产物库选择30种天然产物作为实验药物,包括黄酮类天然产物10种、滋阴养血生津中药单体10种、抗炎抗肿瘤天然产物单体10种。将天然产物单体由DMSO或无水乙醇溶解,配制成50-100mM的药物母液作为储存浓度,使用时,用培养基将母液稀释至合适终浓度,供细胞实验使用。
首先筛选对AQP5启动子具有激活作用的10种黄酮类天然产物;
实验具体如下:用293T细胞以105密度铺48孔板,将荧光素酶报告基因pGL3/AQP5p质粒转染细胞,用1μM的10种黄酮类天然产物作用细胞24小时,应用PBS作为阴性对照,应用0.1μM雌二醇作为阳性对照。双荧光素酶报告基因检测试剂盒进行检测,所得的结果为各个实验样品的荧光素酶的活性与Renilla荧光素酶活性的比值。实验结果均为3次独立的实验结果的平均值,每次实验每组设计3个平行孔,所有的结果均以平均值±标准差(mean±S.D.)表示。
结果如附图1所示,其中9种黄酮类天然产物可激活AQP5启动子,与PBS对照组比较均具有统计学差异。特别是,三种黄酮类天然产物显示了很强的AQP5启动子激活作用,并且显著优于0.1μM雌二醇组对AQP5启动子的转录激活作用。
其中,木犀草素激活AQP5启动子是PBS对照组的约4.2倍,激活作用最为显著。
其次,用相同的实验方法筛选对AQP5启动子具有激活作用的10种滋阴养血生津中药单体;应用PBS作为阴性对照,应用0.1μM雌二醇及1μM木犀草素作为阳性对照。
结果如附图2所示,10种中药单体均可激活AQP5启动子,与PBS对照组比较均具有统计学差异,然而其作用不如木犀草素显著。
用相同的实验方法筛选对AQP5启动子具有激活作用的10种抗炎抗肿瘤天然产物单体;用PBS作为阴性对照,应用0.1μM雌二醇及1μM木犀草素作为阳性对照。
结果如附图3所示,10种抗炎抗肿瘤天然产物单体对AQP5启动子激活作用不显著;其中,2种天然产物对AQP5启动子激活作用与0.1μM雌二醇组类似。
因此,发现显著激活AQP5启动子的天然产物单体木犀草素。
第二步、验证木犀草素上调涎腺细胞AQP5转录水平。
进行Realtime-PCR实验。
用HSG细胞以106密度铺6孔板,用不同浓度木犀草素(0.01μM、0.1μM、1μM)作用细胞48小时,应用0.1μME2作为阳性对照,用TRlzol提取细胞内总RNA,合成cDNA,应用hsAQP5引物(hsAqp5上游引物5-TGCCATCCTTTACTTCTACCTG-3,下游引物5-CTCATACGTGCCTTTGATGATG-3)进行Realtime-PCR。
结果如附图4所示,在HSG细胞中,木犀草素可上调AQP5mRNA水平,且呈剂量依赖性;木犀草素在1μM浓度下作用最为显著,可上调AQP5mRNA水平约为PBS对照组4.7倍。结果证明了木犀草素可上调AQP5转录水平。
第三步、验证木犀草素促进涎腺细胞AQP5蛋白表达水平。
进行蛋白印迹和免疫荧光实验;
用HSG细胞以106密度铺6孔板,用不同浓度木犀草素(0.01μM、0.1μM、1μM)作用细胞48小时,应用0.1μME2作为阳性对照,应用RIPA裂解细胞收蛋白样,westernblot检测在不同浓度木犀草素作用下AQP5蛋白的表达水平。
结果如附图5所示,木犀草素作用下AQP5蛋白水平呈剂量依赖性升高,其中0.1μM、1μM木犀草素显著促进AQP5蛋白表达水平,1μM木犀草素作用最为显著。
用HSG细胞以5×105密度铺12孔板,用1μM木犀草素作用细胞48小时,应用0.1μME2作为阳性对照。
通过免疫荧光染色检测木犀草素对HSG细胞AQP5表达及定位的影响,用AQP5抗体进行免疫荧光染色(红色),细胞核用DAPI染色。
结果如附图6所示,木犀草素作用下HSG细胞AQP5染色强度增加。
以上的结果:证明木犀草素促进AQP5蛋白表达。
第四步、建立去卵巢小鼠动物模型模拟绝经期口干症,分组进行灌胃给药。
建立去卵巢小鼠动物模型,以模拟绝经期口干症。
取雌性ICR小鼠共24只,将小鼠随机分为4组,每组6只,分为假手术/生理盐水组、去卵巢/生理盐水组、去卵巢/木犀草素组、去卵巢/雌二醇组。
首先,对6只小鼠进行假手术,对另外18只小鼠进行去卵巢手术。去卵巢手术小鼠麻醉后在腹部正中做切口,然后切除双侧卵巢,假手术暴露卵巢,移除一小块脂肪组织,切口分层缝合。手术两周后,将去卵巢小鼠进一步按照给药进行分组,小鼠每日接受灌胃给药。将假手术的6只小鼠给予生理盐水(假手术/生理盐水组),将去卵巢的18只小鼠分为3组(每组6只),分别给予生理盐水(去卵巢/生理盐水组)、木犀草素(去卵巢/木犀草素组)、雌二醇(去卵巢/雌二醇组),共给药12周。
需要注意的是,唾液分泌量和饮水量检测是评价小鼠口干症的两种唾液腺功能指标。
当唾液分泌功能障碍时,唾液分泌量减少;饮水量可代表小鼠的口干症状(口干后小鼠会大量饮水)。
因此,检测各组小鼠的唾液分泌量及饮水量,此处请参照附图7,以研究木犀草素对去卵巢小鼠唾液分泌障碍和口干症状的校正效果。在给药前和给药后4周、8周、12周测量小鼠唾液分泌及饮水量,计算各组小鼠唾液分泌指数和周饮水指数的平均值,观察各组之间的变化。唾液分泌指数=棉片重量增加量(mg)/体重(g);周饮水量指数=周饮水量(mL)/体重(g)。给药12周后处死小鼠,取小鼠组织进行组织学染色分析。
第五步、对各组小鼠给药12周的唾液分泌指标进行分析,得出以下结果:木犀草素上调了去卵巢鼠唾液分泌量,校正了去卵巢小鼠的唾液分泌障碍。
在此时,进行比较假手术/生理盐水组(正常对照组)、去卵巢/生理盐水组(疾病组)、去卵巢/木犀草素组(药物组)、去卵巢/雌二醇组(阳性对照组)的4组小鼠给药前和给药后4周、8周、12周的唾液分泌指数,以研究木犀草素对去卵巢小鼠唾液分泌障碍的校正效果。
结果如附图8所示,假手术/生理盐水组(正常对照组)小鼠在各阶段(给药前,给药4周、8周、12周)唾液分泌水平无显著变化,唾液分泌指数在20-25mg/g水平;去卵巢/生理盐水组(疾病组)小鼠给药4周时唾液分泌急剧下降,减少至假手术/生理盐水组一半,给药8-12周唾液分泌下降逐渐缓慢,维持在低水平平台期,12周时去卵巢/生理盐水组(疾病组)小鼠唾液分泌降低至假手术/生理盐水组(正常对照组)的1/3;
去卵巢/木犀草素组(药物组)、去卵巢/雌二醇组(阳性对照组)给药4周时唾液分泌指数较假手术/生理盐水组(正常对照组)显著降低,然而,从给药4周开始,唾液分泌指数开始回升,给药8周后,唾液分泌指数上升并接近至假手术/生理盐水组(正常对照组)水平,给药12周,唾液分泌指数几乎达到假手术/生理盐水组(正常对照组)水平;
说明木犀草素和雌二醇两种药物可显著校正ICR去卵巢小鼠的唾液分泌量,并且在4-8周开始起效。
为了进一步说明结果,对各组小鼠给药12周的唾液分泌指标进行分析,结果如附图9所示:
去卵巢/生理盐水组小鼠(疾病组)唾液分泌指数为假手术/生理盐水组(正常对照组)的1/3,然而,去卵巢/木犀草素组(药物组)、去卵巢/雌二醇组(阳性对照组)唾液分泌指数完全不同于去卵巢/生理盐水组(疾病组),与假手术/生理盐水组(正常对照组)接近。说明木犀草素上调了去卵巢鼠唾液分泌量,校正了去卵巢小鼠的唾液分泌障碍。
第六步、验证木犀草素降低去卵巢小鼠的饮水量,缓解了口干症状。
对假手术/生理盐水组(正常对照组)、去卵巢/生理盐水组(疾病组)、去卵巢/木犀草素组(药物组)、去卵巢/雌二醇组(阳性对照组)的4组小鼠给药前和给药后4周、8周、12周的饮水指数进行比较,以研究木犀草素对去卵巢小鼠口干症状的缓解作用。
结果如附图10所示,假手术/生理盐水组(正常对照组)小鼠在各阶段(给药前,给药4周、8周、12周)饮水指数无显著变化,饮水指数在0.75-1.0mL/g水平;去卵巢/生理盐水组(疾病组)小鼠给药4周时饮水指数上升至假手术/生理盐水组(正常对照组)近1.5倍,给药8-12周饮水指数上升逐渐缓慢,维持在高水平,12周时去卵巢/生理盐水组(疾病组)小鼠饮水指数上升至假手术/生理盐水组(正常对照组)近1.7倍,说明去卵巢后小鼠饮水量显著上升提示其口干症状;
虽然,去卵巢/木犀草素组(药物组)、去卵巢/雌二醇组(阳性对照组)给药4周时饮水指数较假手术/生理盐水组(正常对照组)增加,但是,给药8-12周后,饮水指数下降并接近假手术/生理盐水组(正常对照组)水平,说明木犀草素和雌二醇两种药物可显著降低去卵巢小鼠的饮水量(缓解口干症状),并且在4-8周开始起效。
为了进一步说明结果,对各组小鼠给药12周的饮水指数进行分析,结果如附图11所示:
去卵巢/生理盐水组(疾病组)小鼠周饮水指数约为假手术/生理盐水组(正常对照组)的1.7倍,而去卵巢/木犀草素组(药物组)、去卵巢/雌二醇组(阳性对照组)周饮水指数与假手术/生理盐水组(正常对照组)接近。说明木犀草素降低去卵巢小鼠的饮水量,缓解口干症状。
第七步、验证木犀草素的安全性;
给药12周后处死小鼠,取小鼠心、肺、肝、肾组织进行组织学染色,结果如附图13所示,假手术/生理盐水组、去卵巢/生理盐水组、去卵巢/木犀草素组、去卵巢/雌二醇组小鼠各组织H&E染色未见显著差异,木犀草素是一种天然黄酮类化合物,木犀草素存在于多种植物中,经过实验的小鼠多组织染色未见异常;
补充说明的,如附图12所示,木犀草素主要存在于甘蓝、洋白菜、菜花、甜菜、胡萝卜、芹菜等蔬菜中,葡萄柚、苹果、鳄梨、柠檬等水果中,金银花、菊花、荆芥、夏枯草、洋蓟、紫苏属、黄芩属等天然药材中,此处不进行穷举。
重点说明的,AQP5是唾液分泌关键靶点,AQP5表达降低导致唾液分泌障碍。为调控涎腺细胞水分泌功能,缓解口干症状,本申请以AQP5为靶点,筛选发现激活AQP5的天然产物木犀草素;针对绝经期口干症构建去卵巢小鼠动物模型,通过唾液分泌检测、饮水量检测,发现木犀草素可校正模型小鼠唾液分泌障碍,具有缓解口干症的作用。
综上所述:
1.在细胞水平,木犀草素激活AQP5启动子是PBS对照组的约4.2倍,1μM木犀草素上调AQP5mRNA水平约为PBS对照组4.7倍,并且显著促进AQP5蛋白表达水平。
2.在动物水平,去卵巢/生理盐水组小鼠(疾病组)唾液分泌指数为假手术/生理盐水组(正常对照组)的1/3,然而,去卵巢/木犀草素组(药物组)唾液分泌指数完全不同于去卵巢/生理盐水组(疾病组),与假手术/生理盐水组(正常对照组)接近。说明木犀草素上调了去卵巢鼠唾液分泌量,校正了去卵巢小鼠的唾液分泌障碍。
3.在动物水平,去卵巢/生理盐水组(疾病组)小鼠周饮水指数约为假手术/生理盐水组(正常对照组)的1.7倍,而去卵巢/木犀草素组(药物组)周饮水指数与假手术/生理盐水组(正常对照组)接近;说明木犀草素降低去卵巢小鼠的饮水量,缓解口干症状。
以上显示和描述了本发明的基本原理和主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明要求保护范围由所附的权利要求书及其等效物界定。
Claims (6)
1.一种木犀草素缓解口干症状的方法,其特征在于,包括如下步骤:
第一步、以AQP5启动子为靶点进行双荧光素酶报告基因活性检测的药物筛选;
用293T细胞以105密度铺48孔板,将荧光素酶报告基因pGL3/AQP5p质粒转染细胞,用1μM的10种黄酮类天然产物作用细胞24小时,应用PBS作为阴性对照,应用0.1μM雌二醇作为阳性对照;双荧光素酶报告基因检测试剂盒进行检测,所得的结果为各个实验样品的荧光素酶的活性与Renilla荧光素酶活性的比值;
确认可以显著激活AQP5启动子的天然产物单体木犀草素;
第二步、测试木犀草素上调涎腺细胞AQP5转录水平;
用HSG细胞以106密度铺6孔板,用不同浓度木犀草素(0.01μM、0.1μM、1μM)作用细胞48小时,应用0.1μME2作为阳性对照,用TRlzol提取细胞内总RNA,合成cDNA,应用hsAQP5引物(hsAqp5上游引物5-TGCCATCCTTTACTTCTACCTG-3,下游引物5-CTCATACGTGCCTTTGATGATG-3)进行Realtime-PCR;
第三步、进行蛋白印迹和免疫荧光实验,测试木犀草素促进涎腺细胞AQP5蛋白表达水平;
用HSG细胞以106密度铺6孔板,用不同浓度木犀草素(0.01μM、0.1μM、1μM)作用细胞48小时,应用0.1μME2作为阳性对照,应用RIPA裂解细胞收蛋白样,westernblot检测在不同浓度木犀草素作用下AQP5蛋白的表达水平;
用HSG细胞以5×105密度铺12孔板,用1μM木犀草素作用细胞48小时,应用0.1μME2作为阳性对照;
通过免疫荧光染色检测木犀草素对HSG细胞AQP5表达及定位的影响,用AQP5抗体进行免疫荧光染色(红色),细胞核用DAPI染色;
第四步、建立去卵巢小鼠动物模型模拟绝经期口干症,分组进行灌胃给药;
首先,建立去卵巢小鼠动物模型,模拟绝经期口干症,取雌性ICR小鼠共24只,将小鼠随机分为4组,每组6只,分为假手术/生理盐水组、去卵巢/生理盐水组、去卵巢/木犀草素组、去卵巢/雌二醇组;
其次,对6只小鼠进行假手术,对另外18只小鼠进行去卵巢手术;去卵巢手术小鼠麻醉后在腹部正中做切口,然后切除双侧卵巢,假手术暴露卵巢,移除一小块脂肪组织,切口分层缝合;手术两周后,将去卵巢小鼠进一步按照给药进行分组,小鼠每日接受灌胃给药;将假手术的6只小鼠给予生理盐水(假手术/生理盐水组),将去卵巢的18只小鼠分为3组(每组6只),分别给予生理盐水(去卵巢/生理盐水组)、木犀草素(去卵巢/木犀草素组)、雌二醇(去卵巢/雌二醇组),共给药12周;
最后,以唾液分泌量和饮水量检测评价小鼠口干症唾液腺功能指标;
第五步、对各组小鼠的唾液分泌指标进行分析;
第六步、验证木犀草素降低去卵巢小鼠的饮水量,缓解口干症状;
第七步、在给药12周后,处死小鼠,取小鼠心、肺、肝、肾组织进行组织学染色,验证木犀草素对小鼠各组织无害。
2.根据权利要求1所述的一种木犀草素缓解口干症状的方法,其特征在于:在给药前和给药后4周、8周、12周测量小鼠唾液分泌及饮水量,计算各组小鼠唾液分泌指数和周饮水指数的平均值,观察各组之间的变化。
3.根据权利要求1所述的一种木犀草素缓解口干症状的方法,其特征在于:根据结构比对,从天然产物库选择30种天然产物作为实验药物,包括黄酮类天然产物10种、滋阴养血生津中药单体10种、抗炎抗肿瘤天然产物单体10种;
首先,需要筛选对AQP5启动子具有激活作用的10种黄酮类天然产物;
其次,采用相同的实验方法筛选对AQP5启动子具有激活作用的10种滋阴养血生津中药单体;
最后,采用相同的实验方法筛选对AQP5启动子具有激活作用的10种抗炎抗肿瘤天然产物单体。
4.根据权利要求3所述的一种木犀草素缓解口干症状的方法,其特征在于:其中,实验结果均为3次独立的实验结果的平均值,每次实验每组设计3个平行孔,所有的结果均以平均值±标准差(mean±S.D.)表示。
5.根据权利要求3所述的一种木犀草素缓解口干症状的方法,其特征在于:木犀草素激活AQP5启动子是PBS对照组的约4.2倍,10种中药单体均可激活AQP5启动子,与PBS对照组比较均具有统计学差异;10种抗炎抗肿瘤天然产物单体对AQP5启动子激活作用不显著。
6.根据权利要求3所述的一种木犀草素缓解口干症状的方法,其特征在于:应用PBS作为阴性对照,应用0.1μM雌二醇及1μM木犀草素作为阳性对照。
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