WO2024114832A1 - 黄柏酮在治疗雄激素脱发中的制药用途 - Google Patents

黄柏酮在治疗雄激素脱发中的制药用途 Download PDF

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WO2024114832A1
WO2024114832A1 PCT/CN2024/073635 CN2024073635W WO2024114832A1 WO 2024114832 A1 WO2024114832 A1 WO 2024114832A1 CN 2024073635 W CN2024073635 W CN 2024073635W WO 2024114832 A1 WO2024114832 A1 WO 2024114832A1
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treatment
pyruvate
protein
androgenic alopecia
hair
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金亮
陈杉杉
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上海迪迈和生物技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
    • A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia

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  • the invention belongs to the field of medicines and relates to the pharmaceutical use of pyruvate in treating androgenic alopecia.
  • AGA Androgenetic alopecia
  • AGA is the most common type of hair loss. It is a progressive hair follicle miniaturization disease that begins in adolescence or late adolescence, characterized by the progressive loss of terminal hair on the scalp in a typical distribution. With the changes in people's living habits and dietary structure, the prevalence of androgenic alopecia has increased year by year, and it is showing a trend of younger age. Although hair loss does not affect physical health, it brings serious mental stress and psychological burden to patients. Current studies have shown that androgens are a decisive factor in the onset of AGA; other factors including inflammation around hair follicles, increased life pressure, tension and anxiety, and bad living and eating habits can aggravate the symptoms of AGA.
  • DHT dihydrotestosterone
  • Finasteride is a 5 ⁇ -reductase inhibitor that treats hair loss by reducing the DHT content in the body.
  • Minoxidil is a vasodilator that treats hair loss by improving the microcirculation of hair follicles.
  • Other treatments include laser therapy, hair transplantation, stem cell therapy and other non-drug treatments.
  • these treatments have significant side effects, such as sexual dysfunction, hirsutism, and high costs. Therefore, it is of great significance to find new drugs for the treatment of androgenic alopecia with fewer side effects.
  • Obacunone is mainly derived from plants such as Phellodendron chinense and Dictamni root bark. It is a limonin triterpenoid compound with diverse pharmacological activities. Modern pharmacological studies have shown that obacunone has the following effects: (1) It causes insects to resist feeding; (2) Microtubule inhibition synergistic effect: It can enhance the cytotoxicity of antitumor drugs that inhibit microtubules, but it itself has no cytotoxicity.
  • RNA synthesis inhibitors such as RNA synthesis inhibitors, alkylating factors, and thymidylate synthase inhibitors
  • Protective neuroactivity The methanol extract of Dictamni root bark has a protective effect on nerves.
  • the alcohol extract including obacunone showed strong neuroprotective activity (anti-glutamate-induced neurotoxicity) on primary cultured mouse cortical cells at a concentration of 0.1 ⁇ mol/L;
  • Limonin analogs have multiple biological activities such as anti-tumor, anti-viral, analgesic, anti-inflammatory, and hypnotic.
  • chrysogenum flavone prevents ulcerative colitis in mice by regulating intestinal microbiota, attenuating TLR4/NF- ⁇ B signaling cascades, and improving damaged epithelial barriers; chrysogenum flavone causes sustained expression of MKP-1, thereby inactivating p38MAPK and inhibiting proinflammatory mediators through intracellular MIF; chrysogenum flavone alleviates liver fibrosis by enhancing the antioxidant effect of GPx-4 and inhibiting EMT; chrysogenum flavone and chrysogenum flavone glycosides inhibit human colon cancer (SW480) cells by inducing apoptosis; and through the TGR5 and PPAR ⁇ pathways, drinking water Dietary supplementation with pyruvate stimulated muscle hypertrophy and inhibited hyperglycemia and obesity; it had a strong promoting effect on apoptosis of 22RV1 prostate cancer cells; it alleviated high glucose-induced oxidative damage in
  • the purpose of the present invention is to provide a pharmaceutical use of pyruvate in treating androgenic alopecia in view of the above-mentioned deficiencies in the prior art.
  • the use of pyruvate in the preparation of a drug for inhibiting androgen receptor dimerization to treat androgenic alopecia and improving the hair follicle environment by reducing inflammation and improving the hair follicle vascular microenvironment is preferred.
  • the invention discloses an application of flavonoids in the preparation of drugs for inhibiting androgen receptor dimerization and preventing androgen receptor dimerization from entering the cell nucleus.
  • Androgen receptor is the binding site of dihydrotestosterone in the body. After binding with dihydrotestosterone, androgen receptor will dimerize and enter the nucleus to regulate downstream genes, leading to hair loss. Therefore, preventing androgen receptor dimerization can effectively block the effect of androgen on hair follicle cells, avoid the strong side effects caused by the decrease in androgen content, and play a role in treating androgenic alopecia.
  • the present invention uses computer simulation technology and western blot experiments to prove that huangbai ketone binds to androgen receptor and effectively prevents it from entering the nucleus after dimerization.
  • huangbai ketone has a significant promoting effect on the growth of hair follicles in mice with androgenic alopecia model.
  • the hair density increases significantly and the number of hair follicles and the number of surrounding blood vessels increase significantly.
  • the number of hair follicles returned to a level close to that of the blank group mice without any treatment, and the number of hair follicles in the androgenic alopecia group increased by 40-60%.
  • huangbai ketone can significantly downregulate skin inflammation, which is consistent with the anti-inflammatory activity of huangbai ketone.
  • the vascular microenvironment of mouse skin was improved, and the number and diameter of blood vessels were restored.
  • Figure 1 is the structural diagram of flavonoids
  • Figure 2 is a diagram of the binding pattern of chloroquine and AR (MOE software)
  • Figure 3 is the western blot result of pyruvate inhibiting AR dimerization and nuclear entry
  • Figure 4 is a comparison of photographs of hair changes in animals treated with pyruvate versus those in other groups.
  • Figure 5 is the HE staining picture of the changes of hair follicles over time after treatment with pyruvate
  • Figure 6 is a bar chart showing the number of hair follicles per unit skin in different animal groups
  • Figure 7 shows the diameter of skin blood vessels in different groups of animals (CD31 immunohistochemistry)
  • Figure 8 is a graph showing changes in body weight of animals in each group during the experiment
  • Figure 9 is the result of HE staining of liver and kidney sections after treatment with pyruvate
  • Figure 10 is the results of flow cytometry of single cell suspensions from the skin of animals in different groups to check the inflammation
  • Embodiment 1 is a diagrammatic representation of Embodiment 1:
  • Androgen receptor is the binding site of dihydrotestosterone in the body. After binding with dihydrotestosterone, androgen receptor will dimerize and enter the nucleus to regulate downstream genes, leading to hair loss. Therefore, preventing androgen receptor dimerization can effectively block the effect of androgen on hair follicle cells, avoid the strong side effects caused by the decrease in androgen content, and play a role in treating androgenic alopecia.
  • the structural formula of chappaconone is shown in Figure 1.
  • the MOE software simulated the chappaconone and AR binding site, as shown in Figure 2. It can be seen from Figure 2 that chappaconone can bind to the androgen receptor.
  • Example 2 Western Blot experiments prove that pyruvate can prevent AR dimerization and nuclear entry
  • lNCap culture medium RPMI Medium 1640 (Invitrogen, 11875093) 88ml + FBS (Gibco) 10ml + Glutamax (Invitrogen, 35050) 1ml + Sodium Pyruvate 100mM Solution (Invitrogen, 11360070) 1ml, 37°C, 5% carbon dioxide environment culture for 48h, cells adhere to the wall, and the confluence reaches about 60%.
  • Huangbai ketone stimulation experiment Cell grouping: blank group, dihydrotestosterone control group, Huangbai ketone + dihydrotestosterone group.
  • Huangbai ketone solution was added to the Huangbai ketone + dihydrotestosterone group of adherent cells to make the final concentration of 200 ⁇ mol/L. After 48h of stimulation, dihydrotestosterone mother solution was added to the other two groups except the blank group to make the final concentration of 10-9 M. After 4 hours of stimulation, nuclear protein and plasma protein separation experiments and western Blot experiments were performed.
  • Blocking Prepare 5% skim milk powder with 1 ⁇ TBST solution as blocking solution. Place the membrane in the blocking solution and incubate on a shaker at room temperature for 3 h.
  • Experimental animals reagents: pyruvate (MB6688, Meilun), finasteride (s62514-250m) were first dissolved in DMSO and then prepared with saline to the required concentration.
  • the experimental animals were male 5-week-old C57 mice, weighing 18-22g, with a certificate number of NO.202207119, provided by the Comparative Medicine Center of Yangzhou University.
  • the laboratory room temperature was 25-27°C, the relative humidity was 40%-60%, the ventilation fan was ventilated, the natural light source was 12h/day, and the cages were kept with 5 mice per cage, and the cages were cleaned every three days.
  • mice were randomly divided into 5 groups, 8 in each group, including: blank group (without any treatment), control group, androgenic alopecia group.
  • Group flavonoid treatment group, finasteride treatment group (positive drug group).
  • the three groups of animals in the androgenic alopecia group, flavonoid treatment group, and finasteride treatment group were modeled with androgenic alopecia.
  • 80 mg of testosterone propionate was dissolved in 40 mL of soybean oil to prepare a 2 mg/mL solution, and each mouse was intraperitoneally injected with 0.1 ml/day. After 14 days of continuous intraperitoneal administration, the androgenic alopecia sales model was successful.
  • 0.1 ml of soybean oil was given to the model group every day as a solvent control.
  • the flavonoid treatment group and the finasteride treatment group were treated by gavage.
  • the dose of finasteride was 0.7 mg/kg/day, and the dose of flavonoid was 1.5 mg/kg/day.
  • the control group was gavaged with normal saline every day to eliminate solvent interference.
  • the mice were killed by dislocation of the neck, and the back skin, liver, kidney and blood were taken for subsequent experiments.
  • Example 4 Flow cytometry to identify skin inflammation
  • Instruments and reagents flow cytometer (BD FACSCeleta), F4/80 (ab204266), CD86 (ab242142).

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Abstract

一种黄柏酮在制备治疗雄激素性脱发的药物中的应用。黄柏酮可抑制AR二聚化治疗雄激素脱发，同时还可通过降低炎症和改善毛囊血管微环境改善毛囊环境。因此，黄柏酮可以在制备治疗雄激素性脱发的药物中应用。

Description

黄柏酮在治疗雄激素脱发中的制药用途 技术领域
本发明属于药物领域，涉及黄柏酮在治疗雄激素脱发中的制药用途。
背景技术
雄激素性脱发(androgenetic alopecia，AGA)是一种最常见的脱发类型，是起始于青春期或青春后期的一种进行性毛囊微小化的脱发疾病，特征是头皮上的末端毛发呈典型分布的渐进性脱落。随着人们生活习惯和饮食结构的改变，雄激素性脱发的患病率逐年增多，并呈现低龄化趋势。脱发虽不影响身体健康，但给患者带来了严重的精神压力和心理负担。目前的研究表明，雄激素在AGA的发病中占有决定性因素；其他包括毛囊周围炎症、生活压力的增大、紧张和焦虑、不良的生活和饮食习惯等因素均可加重AGA的症状。脱发区毛囊内雄激素受体基因表达升高和/或Ⅱ型5α还原酶基因表达升高，从而导致雄激素对易感毛囊的作用增大。对于AGA而言，易感毛囊中真皮成分细胞内含有特定的Ⅱ型5α还原酶，可以将血液中循环至该区域的雄激素睾酮转化为二氢睾酮(DHT)，通过与细胞内的雄激素受体结合引起一系列反应，进而使毛囊出现进展性的微型化和脱发直至秃发。
截止目前，对于雄激素脱发治疗经过FDA批准的药物只有两种，为口服药物非那雄胺和外用涂抹剂米诺地尔。非那雄胺为5α还原酶抑制剂，通过减少体内DHT含量治疗脱发。米诺地尔为血管扩张剂，通过改善毛囊微循环，达到治疗脱发的效果。其他治疗手段包括激光治疗，毛发移植，干细胞治疗等非药物治疗手段。但是这些治疗方法都存在很大的副作用，如性功能障碍，多毛症，费用高等问题。因此寻找新的且副作用更小的治疗雄激素脱发药物具有重要意义。
黄柏酮(Obacunone)主要来源于黄柏、白鲜皮等植物，为柠檬苦素类三萜化合物，具有多样药理活性。现代药理研究表面，黄柏酮有以下作用：(1)使昆虫产生拒食行为；(2)微管抑制增效作用：能增强具有抑制微管作用的抗肿瘤药的细胞毒性，其本身没有细胞毒性。而对其他抗肿瘤药物如RNA合成抑制剂、烷化剂因子、胸苷酸合成酶抑制剂等没有显示出明显的增效作用；(3)保护神经活性：白鲜根皮的甲醇提取物对神经有保护作用。包括黄柏酮在内的醇提物在0.1μmol/L浓度时，对原始培养的老鼠皮质细胞表现出很强的神经保护活性(抗谷氨酸盐诱导的神经毒性)；(4)柠檬苦素类似物具有抗肿瘤、抗病毒、镇痛、抗炎、催眠等多种生物活性。有研究报道：黄柏酮通过调节肠道微生物群、减弱TLR4/NF-κB信号级联反应和改善受损的上皮屏障来预防小鼠溃疡性结肠炎；黄柏酮引起MKP-1的持续表达，从而使p38MAPK失活，通过细胞内MIF抑制促炎介质；黄柏酮通过增强GPx-4的抗氧化作用和抑制EMT来减轻肝纤维化；黄柏酮和黄柏酮糖苷通过诱导凋亡抑制人结肠癌(SW480)细胞；通过TGR5和PPARγ途径，饮食补充黄柏酮刺激肌肉肥大，抑制高血糖和肥胖；对22RV1前列腺癌细胞凋亡有很强的促进作用；通过抑制GSK-3β活性减轻高糖诱导的NRK-52E细胞氧化损伤；通过抑制MCF-7人乳腺癌细胞中的p38MAPK信号通路，黄柏酮在体外表现出抗增殖和抗芳香化酶活性；通过激活NRF2延缓常染色体显性多囊肾病患者的肾囊肿发展；减少慢性炎症诱导的结肠直肠癌小鼠的炎症信号和肿瘤发生。
尽管黄柏酮作为药物组分之一应用已久，但未见报道黄柏酮治疗雄激素脱发的作用。
发明内容
本发明的目的是针对现有技术的上述不足，提供黄柏酮在治疗雄激素脱发中的制药用途。
本发明的目的可通过以下技术方案实现：
黄柏酮在制备治疗雄激素性脱发的药物中的应用。
作为本发明的一种优选，黄柏酮在制备抑制雄激素受体二聚化治疗雄激素脱发、通过降低炎症和改善毛囊血管微环境改善毛囊环境的药物中的应用。
黄柏酮在制备抑制雄激素受体二聚化、阻止雄激素受体二聚化进入细胞核的药物中的应用。
有益效果：
雄激素受体是二氢睾酮在体内的结合位点，与二氢睾酮结合后，雄激素受体会发生二聚化并且入核调控下游基因，导致毛发脱落。因此阻止雄激素受体二聚化可有效阻断雄激素对毛囊细胞的作用，可避免雄激素含量下降导致较强副作用的同时，起到治疗雄激素脱发的作用。本发明通过计算机模拟技术和western blot实验证明黄柏酮与雄激素受体结合有效阻止其二聚化后入核。本发明实验研究发现黄柏酮对雄激素脱发模型小鼠毛囊生长就有明显的促进作用。一般在给药21天后毛发密度明显增加且毛囊数量和周围血管数量显著增加。以灌胃给药的方式以1.5mg/kg/d给药21天后，毛囊数量恢复到接近未经任何处理的空白组小鼠，对比雄激素脱发组毛囊数量增加40-60％。通过流式细胞术对小鼠皮肤细胞悬液进行分析后发现，黄柏酮可明显下调皮肤炎症，这也黄柏酮抗炎活性相吻合。在黄柏酮给药21天后，小鼠皮肤血管微环境得以改善，血管数量和直径都得以恢复。
经过对小鼠体内二氢睾酮测量发现黄柏酮并未降低小鼠体内二氢睾酮含量，肝肾切片并未发现明显病变，表明黄柏酮无细胞毒性，对肾脏细胞活力没有显著影响。
本实验结果提示，黄柏酮可抑制AR二聚化治疗雄激素脱发，同时还可通过降低炎症和改善毛囊血管微环境改善毛囊环境。
附图说明
图1是黄柏酮结构图
图2是黄柏酮与AR结合模式图(MOE软件)
图3是黄柏酮抑制AR二聚化入核western blot结果图
图4是黄柏酮治疗与其他组动物毛发变化照片对比图。
图5是黄柏酮治疗后毛囊随时间变化HE染色图片
图6是不同动物组别单位皮肤毛囊数量统计条形图
图7是不同组别动物皮肤血管直径情况(CD31免疫组化)
图8是实验过程中各组动物体重变化图
图9是黄柏酮治疗后肝肾切片HE染色结果图
图10是不同组别动物皮肤单细胞悬液流式细胞术查看炎症情况结果图
具体实施方式
实施例1：
雄激素受体是二氢睾酮在体内的结合位点，与二氢睾酮结合后，雄激素受体会发生二聚化并且入核调控下游基因，导致毛发脱落。因此阻止雄激素受体二聚化可有效阻断雄激素对毛囊细胞的作用，可避免雄激素含量下降导致较强副作用的同时，起到治疗雄激素脱发的作用。黄柏酮结构式见图1，经MOE软件模拟黄柏酮和AR结合位点，见图2。由图2可见黄柏酮能够与雄激素受体结合。
实施例2：Western Blot实验证明黄柏酮可阻止AR二聚化入核
1.实验仪器：细胞培养瓶、培养皿(NEST公司)，Trans-Blot半干转系统(Bio-Rad)，化学发光成像仪(上海天能科技有限公司)，蛋白电泳槽(Bio-Rad)2.实验耗材、试剂和细胞信息：蛋白胶预混液(E302-01,E303-01诺唯赞生物有限公司)，细胞核蛋白与细胞浆蛋白抽提试剂盒(P0027,碧云天)，RIPA强效蛋白裂解液(P0013B，碧云天)，雄激素受体一抗(A19611,ABclonal)二氢睾酮(CHB16A802100G)，黄柏酮(MB6688,美仑)，Lncap细胞系(样本编号为：20210106-06，生物安全等级：BSL-1，厦门逸漠生物科技有限公司)，RPMI Medium1640(Invitrogen,11875093)，FBS(Gibco)，Glutamax(Invitrogen,35050)，Sodium Pyruvate 100mM Solution(Invitrogen,11360070)PBS ph7.4basic(C10010500BT),TBST(实验室配置)，DMSO(D2650,Sigma),无水乙醇(大茂)
3.实验方法与结果：
3.1黄柏酮溶液配置：DMSO溶解后浓度为4.4*105μmol/L作为母液备用。配置10-6M二氢睾酮溶液：将二氢睾酮溶液无水乙醇中配置10-2M溶液，经培养基梯度稀释至10-6M备用。
3.2LNcap细胞培养：配置lNCap培养基：RPMI Medium 1640(Invitrogen,11875093)88ml+FBS(Gibco)10ml+Glutamax(Invitrogen,35050)1ml+Sodium Pyruvate 100mM Solution(Invitrogen,11360070)1ml，37℃，5％二氧化碳环境培养48h，细胞贴壁，汇合度达到60％左右。黄柏酮刺激实验：细胞分组情况：空白组，二氢睾酮对照组，黄柏酮+二氢睾酮组。首先向贴壁细胞中黄柏酮+二氢睾酮组加入黄柏酮溶液，使得终浓度为200μmol/L,刺激48h后，向除了空白组另外两组中加入二氢睾酮母液，使得终浓度为10-9M,刺激4小时后，进行细胞核蛋白与浆蛋白分离实验和western Blot实验。
3.3细胞核蛋白与浆蛋白分离实验步骤：
(1)准备溶液：室温融解试剂盒中的细胞浆蛋白抽提试剂A；细胞浆蛋白抽提试剂B；细胞核蛋白抽提试剂，溶解后立即放置在冰上，混匀。取适当量的细胞浆蛋白抽提试剂A备用，在使用前数分钟内加入PMSF，使PMSF的最终浓度为1mM。
(2)对于贴壁细胞：用PBS洗一遍，用细胞刮子刮下细胞，并用移液器吹打下细胞。离心收集细胞，吸尽上清，留下细胞沉淀备用。
每20微升细胞沉淀加入200微升添加了PMSF的细胞浆蛋白抽提试剂A。
(3)最高速剧烈震荡5秒，把细胞沉淀完全悬浮并分散开。(如果细胞沉淀没有完全悬浮并分散开，可以适当延长vortex时间。
(4)冰浴15分钟。
(5)加入细胞浆蛋白抽提试剂B10微升。最高速剧烈震荡5秒，冰浴1分钟。(6)高速 剧烈震荡5秒，4℃12,000g离心5分钟。
(7)对于沉淀，完全吸尽残余的上清，加入50微升添加了PMSF的细胞核蛋白抽提试剂。
(8)高速剧烈震荡20秒，把细胞沉淀完全悬浮并分散开。然后放回冰浴中，每隔2分钟再高速剧烈震荡20秒，共30分钟。
(9)4℃12,000g离心10分钟。
3.4Western blot
(1)分离胶制备。将玻璃板清洗干净晾干后放入制胶模具中，加入蒸馏水检漏，检查完毕后使用干燥滤纸将水分吸出。根据所用蛋白大小，AR大小110KDa，选择8％的蛋白胶配置。使用移液器将液体分离胶缓慢加入制胶模具中，避免产生气泡，加入1mL异丙醇后静置20min等待分离胶凝固。
(2)浓缩胶制备。待分离胶完全凝固后，弃去上层异丙醇溶液，使用ddH2O润洗三次，洗涤后用干燥滤纸将残留水分吸出后可进行浓缩胶的制备。
(3)蛋白上样。将-20℃保存的蛋白样品于冰上融化；待浓缩胶凝固后，将胶板安装到电泳槽，并在槽内加满电泳缓冲液，轻轻竖直拔出齿梳。选择合适的泳道加入5μL蛋白Marker和30μg蛋白样品，加样时对蛋白样进行涡旋，保证蛋白的均匀分散。
(4)蛋白电泳。先使用60V恒压电泳30min，接着调整电压至120V，直到蛋白marker跑出清晰的条带，溴酚蓝跑至接近泳道底端，约60min。
(5)蛋白转膜(半干转)。将滤纸置于预冷的1×转膜缓冲液(Turbo)中浸泡。，裁剪尺寸为4.5cm*8.5cm大小PVDF膜浸泡在甲醇中活化5min。电泳结束后，取出电泳胶板，将胶切至适当大小。按自下而上分别是滤纸-PVDF膜-凝胶-滤纸的顺序放置。该过程需要注意放置每层时都需要避免气泡的出现，并且使凝胶保持湿润状态。转膜时间为30min。
(6)封闭。用1×TBST溶液配置5％脱脂奶粉作为封闭液。将膜放置于封闭液中，室温摇床孵育3h。
(7)一抗孵育。弃去封闭液，用1×TBST摇床洗涤3次，每次10min。将PVDF膜进行适当的裁剪，做好标记后加入相应的一抗，4℃孵育过夜。一抗用含5％BSA的TBST溶液按1:1000的稀释比稀释。
(8)二抗孵育。吸弃一抗后，用1×TBST摇床洗涤3次，每次10min。二抗用1×TBST溶液按1:10000的比例稀释，室温摇床孵育45min。
(9)曝光与成像分析。孵育结束后吸弃二抗，用1×TBST摇床洗涤3次，每次10min。ECL发光液现用现配，均匀覆盖在蛋白膜上，并用凝胶成像仪对蛋白条带进行显影分析。
结果显示：通过蛋白条带可明显看出经过黄柏酮刺激的细胞，细胞核内AR量明显减少，证明AR可有效阻止AR入核发挥作用(图3)。
实施例3.雄激素脱发动物模型的构建及体内药物活性验证
1.实验动物，试剂：黄柏酮(MB6688,美仑)，非那雄胺(s62514-250m)两种药物均用时先用DMSO将其溶解后，以生理盐水配制成所需浓度。试验动物为雄性5周龄C57小鼠，体重18-22g，合格证号：NO.202207119，由扬州大学比较医学中心提供。实验室室温25-27℃，相对湿度40％-60％，换气扇通气，自然光源12h/日，笼养，每笼5只，每三日清扫笼舍一次。
2.取小鼠随机分为5组，每组8只，为:空白组(未经任何处理)，对照组，雄激素脱发 组，黄柏酮治疗组，非那雄胺治疗组(阳性药物组)。首先对雄激素脱发组，黄柏酮治疗组，非那雄胺治疗组三组动物进行雄激素脱发造模，将80mg丙酸睾酮溶解于40mL大豆油配置成2mg/mL的溶液,每只小鼠腹腔注射0.1ml/天。连续腹腔给药14天雄激素脱发销售模型成功。同时每天给予模型组0.1ml大豆油作为溶剂对照。14天后开始对黄柏酮治疗组和非那雄胺治疗组采用灌胃的给药方式进行治疗，非那雄胺给药剂量为0.7mg/kg/天，黄柏酮给药剂量为1.5mg/kg/天，对照组每天灌胃生理盐水排除溶剂干扰。连续给药21天后脱颈处死老鼠，分别取背部皮肤，肝脏，肾脏和血液进行后续实验。皮肤，肝脏，肾脏取部分放于4％多聚甲醛中固定，用于后续HE染色和免疫组化实验，其余组织放在-80℃冰箱备用。血液四度静置24h后取上层血清放置-80℃备用。
结果：与模型组相比，HE组织切片染色显示黄柏酮治疗组的毛囊数量明显增加(图5、6)，CD31免疫组化显示经过治疗毛囊周围血管直径增加改善血管退行化现象(图7)。治疗效果好于非那雄胺阳性药物组。而肝脏切片结果未见明显损伤(图9)，表明黄柏酮治疗脱发上的安全性高，因此本次结果显示，黄柏酮可以逆转雄激素介导的雄激素脱发损伤，可治疗雄激素脱发，恢复毛发的生长。
实施例4流式细胞术鉴定皮肤炎症情况
1.仪器和试剂：流式细胞仪(BD FACSCeleta)，F4/80(ab204266),CD86(ab242142)。
2.实验步骤：获取新鲜皮肤后，用剪刀剪碎，利用一型胶原酶37℃水浴消化30min后，终止消化后1600r离心5min弃上清，加入0.25％胰酶进行消化，37℃消化30min，离心后，利用细胞筛过滤，去除组织块，获得皮肤单细胞悬液，将各组细胞分别加入染F4/80和CD86荧光抗体，避光室温染色30min后。利用流式细胞术进行实验。
实验结果显示：黄柏酮可明显下调睾酮带来的皮肤炎症(图10)。

Claims (3)

  1. 黄柏酮在制备治疗雄激素性脱发的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于黄柏酮在制备抑制雄激素受体二聚化治疗雄激素脱发、通过降低炎症和改善毛囊血管微环境改善毛囊环境的药物中的应用。
  3. 黄柏酮在制备抑制雄激素受体二聚化、阻止雄激素受体二聚化进入细胞核的药物中的应用。
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