(Z)-Guggulsterone   中文名称: 香胶甾酮Z

VEGF-R2

在 HUVEC 中，(Z)-GugguLsterone（10、20 μM；24 或 48 小时）可降低 VEGF-R2 蛋白水平 [1]。通过 FXR 介导的 ACE2 调节，(Z)-Guggulsterone (10 μM；24) 可减少初级气道，扰乱类器官中的 ACE2 和 SHP 水平，并减少许多细胞类型中的 SARS-CoV-2 感染 [2]。

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z-guggulsterone治疗以浓度和时间依赖的方式抑制人脐静脉内皮细胞（HUVEC）的毛细血管样管形成（体外新生血管形成）以及HUVEC和DU145人前列腺癌症细胞的迁移。guggulsterone的z和E异构体作为HUVEC管形成的抑制剂似乎是等效的[1]。

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前期研究发现，z-guggulsterone是印度阿育吠陀药用植物commiora mukul的一种成分，通过引起细胞凋亡抑制人前列腺癌细胞的生长。我们现在报道了一种新的对z-guggulsterone的反应，涉及体外和体内血管生成的抑制。z-谷固酮治疗抑制人脐静脉内皮细胞（HUVEC）的毛细血管样管形成（体外新生血管），并以浓度和时间依赖性方式抑制HUVEC和DU145人前列腺癌细胞的迁移。谷谷固酮的z-和e -异构体似乎具有抑制HUVEC管形成的同等效力。z-guggulsterone介导的体外血管生成抑制与抑制促血管生成生长因子（如血管内皮生长因子（VEGF）和粒细胞集落刺激因子）的分泌、下调VEGF受体2 （VEGF- r2）蛋白水平和Akt失活相关。z-guggulsterone介导的对DU145细胞迁移的抑制通过敲低VEGF-R2蛋白水平而增强。在DU145细胞中，组成活性Akt的异位表达对z-guggulsterone介导的细胞迁移抑制[1]具有保护作用。

(Z)-Guggulsterone（二氧化硅；1 mg；每周 5 次）可显着降低湿重和肿瘤体积 [1]。
在DU145 Matrigel塞试验中，对雄性裸鼠口服1mg z-guggulsterone/d（五次/周）抑制了体内血管生成，其证据是肿瘤负荷、微血管面积（血管生成标志物因子VIII和CD31染色）和VEGF-R2蛋白表达在统计学上显著降低。总之，本研究表明，z-guggulsterone通过抑制VEGF-VEGF-R2-Akt信号轴来抑制血管生成。总之，我们的研究结果为z-guggulsterone对前列腺癌症的疗效的进一步临床前和临床研究提供了令人信服的理由[1]。

z-guggulsterone介导的体外血管生成抑制与抑制促血管生成生长因子[如血管内皮生长因子（VEGF）和粒细胞集落刺激因子]的分泌、下调VEGF受体2（VEGF-R2）蛋白水平和Akt失活相关。z-guggulsterone介导的对DU145细胞迁移的抑制通过敲低VEGF-R2蛋白水平而增加。DU145细胞中组成型活性Akt的异位表达对z-guggulsterone介导的细胞迁移抑制具有保护作用[3]。

蛋白质印迹分析 [1]
细胞类型：血管内皮生长因子 (VEGF)
测试浓度： 10、20 μM
孵育持续时间：24 或 48 小时
实验结果：导致 HUVEC 中 VEGF-R2 蛋白水平下降。

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细胞培养与细胞活力检测 [1]
人脐静脉内皮细胞（HUVEC）购自Clonetics公司，培养于添加5%胎牛血清的内皮细胞生长培养基-2（EGM2 MV SingleQuots）中。DU145细胞单层培养方法如我们先前所述。香胶甾酮各异构体的母液用DMSO配制，并用完全培养基稀释。对照组加入等体积DMSO（终浓度<0.2%）。Z型和E型香胶甾酮对HUVEC活力的影响通过磺酰罗丹明B法测定，方法如我们先前所述。

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体外毛细血管样管腔形成与迁移实验 [1]
Z型和E型香胶甾酮对体外血管生成的影响通过管腔形成实验评估，方法如我们先前报道。接种于Matrigel的HUVEC可分化形成毛细血管样管状结构。部分管腔形成实验中，HUVEC在存在或不存在1 μmol/L Akt-1/2抑制剂条件下，暴露于20 μmol/L Z型香胶甾酮处理24小时。采用Transwell Boyden小室（聚碳酸酯滤膜孔径8 μm）评估Z型香胶甾酮对HUVEC或DU145细胞体外迁移的影响，方法如我们先前所述。部分迁移实验中，HUVEC或DU145细胞在存在或不存在1 μmol/L Akt-1/2抑制剂条件下，用20 μmol/L Z型香胶甾酮处理24小时。

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免疫印迹分析 [1]
总Akt、磷酸化Akt及VEGF-R2的免疫印迹检测方法如我们先前所述。简言之，用指定浓度Z型香胶甾酮处理HUVEC或DU145细胞特定时间后，收集悬浮与贴壁细胞。细胞裂解液制备方法如前述。裂解蛋白经6%-10% SDS-PAGE分离后转印至聚偏二氟乙烯膜。经一抗和二抗处理后，采用增强化学发光法显色免疫反应条带。剥离印迹膜后用抗肌动蛋白抗体复染以校正上样量差异。通过条带光密度扫描定量蛋白水平变化。每种蛋白的免疫印迹实验均使用独立制备的裂解液重复至少两次。

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生长因子、白细胞介素及基质金属蛋白酶分析 [1]
将HUVEC或DU145细胞（2×10⁵个）接种于24孔板，孵育过夜使贴壁。细胞用指定浓度Z型香胶甾酮或DMSO（对照）处理24及48小时后，收集培养上清，采用商品化ELISA试剂盒检测VEGF、EGF、G-CSF、FGF、IL-12、IL-17、MMP-2和MMP-9的分泌水平，方法如我们先前所述。

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VEGF-R2的RNA干扰 [1]
采用靶向VEGF-R2的小干扰RNA（siRNA）进行基因沉默。非特异性对照siRNA购自Qiagen公司。转染时，将DU145细胞（5×10⁴个）接种于6孔板过夜贴壁，按制造商推荐方案使用OligofectAMINE转染200 nmol/L对照siRNA或VEGF-R2靶向siRNA。转染24小时后，用DMSO（对照）或20 μmol/L Z型香胶甾酮处理细胞24小时，收集细胞进行迁移实验和免疫印迹分析，方法同前。

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组成型活性Akt的异位表达 [1]
采用Fugene 6转染试剂将编码组成型活性Akt-1（Myr-Akt1-HA）的pCMV6载体或空载体瞬时转染DU145细胞。简言之，将DU145细胞以2×10⁵个/mL密度接种于6孔板过夜贴壁，转染组成型活性Akt表达载体或空载体。转染24小时后，用20 μmol/L Z型香胶甾酮或DMSO（对照）处理细胞24小时，检测总Akt/磷酸化Akt水平并进行迁移实验。

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‌染色质免疫沉淀(ChIP)实验 [2]‌
每组ChIP实验使用约6×10⁶个细胞。细胞在收集前2小时用含100 μM CDCA(鹅去氧胆酸)、UDCA(熊去氧胆酸)或Z型香胶甾酮(ZGG)的新鲜培养基孵育。使用True Micro ChIP试剂盒按说明书操作：1)预清除后，裂解液与FXR抗体(附表1)或非免疫IgG 4℃孵育过夜；2)通过MicroChip DiaPure柱纯化免疫沉淀DNA；3)采用ΔΔCt法进行qPCR分析(引物序列见附表3)。阳性对照使用FXR靶基因OSTα(即SLC51A)FXRE侧翼引物，阴性对照使用ACE2启动子非FXRE区域引物。结果以非免疫IgG ChIP对照的富集程度进行标准化。

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‌荧光素酶报告基因实验 [2]‌
以人类基因组DNA为模板，扩增ACE2基因和SHP基因(即NR0B2)中FXRE IR-1片段，克隆至pGL3-promoter荧光素酶载体。通过定点突变构建ACE2和SHP IR-1突变体(引物序列见附表4)。使用TransIT-293转染试剂将报告基因载体与FXR表达质粒共转染HEK293细胞。转染24小时后，用50 μM CDCA、UDCA或ZGG处理8小时。采用GLO-Luciferase Reporter系统检测荧光素酶活性，结果以空pGL3载体标准化。

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‌细胞毒性与活力检测 [2]‌
类器官分别用0.1-100 μM CDCA、UDCA或ZGG处理：1)台盼蓝染色后用Countess II细胞计数仪计数活细胞百分比；2)10 μM处理组采用PrestoBlue刃天青法，使用SpectraMax M2酶标仪(SoftMax Pro 5.4.4软件)检测细胞活力。

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‌SARS-CoV-2复制荧光素酶报告系统 [2]‌
构建方法如文献28所述：1)HEK293T报告细胞稳定表达ACE2、海肾荧光素酶(Rluc)和SARS-CoV-2木瓜样蛋白酶激活的环状排列萤火虫荧光素酶(FFluc)；2)接种于96孔板后，用不同浓度CDCA、UDCA或ZGG处理，以MOI=0.01感染SARS-CoV-2；3)阳性对照为瑞德西韦(RdRp抑制剂)和REGN-COV2(中和抗体组合)；4)24小时后裂解细胞，采用Dual-Glo试剂盒测定FFluc/Rluc活性比，通过GraphPad Prism软件进行4参数logistic曲线拟合分析。

Animal/Disease Models: Male nude mice (5-6 weeks old) were subcutaneously (sc) (sc) implanted with Matrigel plugs containing DU145 cells.
Doses: 1 mg.
Route of Administration: po (po (oral gavage)) 5 times a week.
Experimental Results: Resulting in statistically significant tumor volume and wet tumor weight. reduce.

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In vivo Matrigel Plug Assay [1]
The effect of z-guggulsterone administration on in vivo angiogenesis was determined by DU145-Matrigel plug assay. Male nude mice (5–6 weeks old) were purchased from Taconic and randomized into two groups of five mice per group. The mice were orally gavaged with 0.1 mL of vehicle (PBS) or 1 mg of z-guggulsterone/mouse in 0.1 mL of PBS (corresponding to ∼40 mg z-guggulsterone/kg body weight) five times per week for 2 weeks prior to Matrigel plug implantation. The Matrigel plugs containing 3 × 106 DU145 cells were implanted s.c. into the flank of each mouse. The z-guggulsterone and vehicle administration was continued for two more weeks. Tumor volume was determined by using a caliper as we have previously described. Body weights of the vehicle-treated control and z-guggulsterone–treated mice were recorded weekly. Mice from each group were also monitored for other symptoms of side effects, including food and water withdrawal and impaired posture or movement. Animals were sacrificed 14 days after Matrigel plug implantation. At the termination of the experiment, the Matrigel plugs from control and z-guggulsterone–treated mice were removed and fixed in 10% neutral-buffered formalin. The fixed Matrigel plugs from control and z-guggulsterone administered mice were dehydrated, embedded in paraffin, and sectioned at 4 μm of thickness. Sections from control and z-guggulsterone administered mice were used for immunohistochemical analysis of CD31, factor VIII, and VEGF-R2. Quantitative image analysis of the microvessel area based on CD31 and factor VIII immunostaining was done using Image Analysis software.

[1]. z-Guggulsterone, a constituent of Ayurvedic medicinal plant Commiphora mukul, inhibits angiogenesis in vitro and in vivo. Mol Cancer Ther. 2008 Jan;7(1):171-80.

[2]. FXR inhibition may protect from SARS-CoV-2 infection by reducing ACE2. Nature. 2023 Mar;615(7950):134-142.

Guggulsterone is a 3-hydroxy steroid. It has a role as an androgen.
Guggulsterone has been reported in Commiphora mukul and Commiphora wightii with data available.

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Our previous studies have shown that z-guggulsterone, a constituent of Indian Ayurvedic medicinal plant Commiphora mukul, inhibits the growth of human prostate cancer cells by causing apoptosis. We now report a novel response to z-guggulsterone involving the inhibition of angiogenesis in vitro and in vivo. The z-guggulsterone treatment inhibited capillary-like tube formation (in vitro neovascularization) by human umbilical vein endothelial cells (HUVEC) and migration by HUVEC and DU145 human prostate cancer cells in a concentration- and time-dependent manner. The z- and E-isomers of guggulsterone seemed equipotent as inhibitors of HUVEC tube formation. The z-guggulsterone-mediated inhibition of angiogenesis in vitro correlated with the suppression of secretion of proangiogenic growth factors [e.g., vascular endothelial growth factor (VEGF) and granulocyte colony-stimulating factor], down-regulation of VEGF receptor 2 (VEGF-R2) protein level, and inactivation of Akt. The z-guggulsterone-mediated suppression of DU145 cell migration was increased by knockdown of VEGF-R2 protein level. Ectopic expression of constitutively active Akt in DU145 cells conferred protection against z-guggulsterone-mediated inhibition of cell migration. Oral gavage of 1 mg z-guggulsterone/d (five times/wk) to male nude mice inhibited in vivo angiogenesis in DU145-Matrigel plug assay as evidenced by a statistically significant decrease in tumor burden, microvessel area (staining for angiogenic markers factor VIII and CD31), and VEGF-R2 protein expression. In conclusion, the present study reveals that z-guggulsterone inhibits angiogenesis by suppressing the VEGF-VEGF-R2-Akt signaling axis. Together, our results provide compelling rationale for further preclinical and clinical investigation of z-guggulsterone for its efficacy against prostate cancer.[1]

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Preventing SARS-CoV-2 infection by modulating viral host receptors, such as angiotensin-converting enzyme 2 (ACE2)1, could represent a new chemoprophylactic approach for COVID-19 that complements vaccination2,3. However, the mechanisms that control the expression of ACE2 remain unclear. Here we show that the farnesoid X receptor (FXR) is a direct regulator of ACE2 transcription in several tissues affected by COVID-19, including the gastrointestinal and respiratory systems. We then use the over-the-counter compound z-guggulsterone and the off-patent drug ursodeoxycholic acid (UDCA) to reduce FXR signalling and downregulate ACE2 in human lung, cholangiocyte and intestinal organoids and in the corresponding tissues in mice and hamsters. We show that the UDCA-mediated downregulation of ACE2 reduces susceptibility to SARS-CoV-2 infection in vitro, in vivo and in human lungs and livers perfused ex situ. Furthermore, we reveal that UDCA reduces the expression of ACE2 in the nasal epithelium in humans. Finally, we identify a correlation between UDCA treatment and positive clinical outcomes after SARS-CoV-2 infection using retrospective registry data, and confirm these findings in an independent validation cohort of recipients of liver transplants. In conclusion, we show that FXR has a role in controlling ACE2 expression and provide evidence that modulation of this pathway could be beneficial for reducing SARS-CoV-2 infection, paving the way for future clinical trials.[2]

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Tumor angiogenesis (neovascularization) is a highly complex process that is regulated by multiple proangiogenic growth factors and their corresponding receptors. Based on the results of the present study, it seems reasonable to conclude that inhibition of the VEGF–VEGF-R2–Akt signaling axis may be an important mechanism in the antiangiogenic effects of z-guggulsterone. This conclusion is supported by the following observations: (a) z-guggulsterone–mediated inhibition of tube formation and migration correlates with the suppression of secretion of VEGF, which provides prosurvival signals to normal and tumor-derived endothelial cells mediated by receptor tyrosine kinase VEGF-R2; (b) z-guggulsterone treatment down-regulates the protein levels of VEGF-R2; (c) z-guggulsterone–mediated suppression of DU145 cell migration is intensified by the knockdown of VEGF-R2 protein levels; (d) z-guggulsterone inhibits Akt in HUVEC and DU145 cells and inhibition of HUVEC tube formation by this agent is intensified by pharmacologic inhibition of Akt. However, the precise mechanism by which z-guggulsterone reduces the secretion of VEGF or down-regulates VEGF-R2 protein level is not clear and requires further investigation. In conclusion, the present study reveals that z-guggulsterone inhibits angiogenesis in vitro and in vivo. The z-guggulsterone–mediated inhibition of angiogenesis is associated with the inactivation of Akt, suppression of growth factor (VEGF and G-CSF), IL-17 and MMP-2 secretion, and down-regulation of VEGF-R2 protein expression.[1]

C21H28O2

312.45

312.208

C, 80.73; H, 9.03; O, 10.24

39025-23-5

39025-23-5

6450278

White to off-white solid powder

1.1±0.1 g/cm3

463.3±45.0 °C at 760 mmHg

188-190°

172.3±25.7 °C

0.0±1.1 mmHg at 25°C

1.557

3.65

34.14

0

2

0

23

640

5

C/C=C/1\C(=O)C[C@H]2[C@@H]3CCC4=CC(=O)CC[C@]4(C)[C@H]3CC[C@]12C

WDXRGPWQVHZTQJ-OSJVMJFVSA-N

InChI=1S/C21H28O2/c1-4-16-19(23)12-18-15-6-5-13-11-14(22)7-9-20(13,2)17(15)8-10-21(16,18)3/h4,11,15,17-18H,5-10,12H2,1-3H3/b16-4+/t15-,17+,18+,20+,21-/m1/s1

(8R,9S,10R,13S,14S,17Z)-17-ethylidene-10,13-dimethyl-1,2,6,7,8,9,11,12,14,15-decahydrocyclopenta[a]phenanthrene-3,16-dione

Z-Guggulsterone; (Z)-Guggulsterone; Z-Guggulsterone; Guggulsterone; 39025-23-5; 95975-55-6; Guggulsterones Z; Cis-Guggulsterone; Guggulsterone E&Z; (Z)-Guggulsterone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO: 5~10 mg/mL (16.0~32.0 mM)
Ethanol: ~2 mg/mL (~6.4 mM)

配方 1 中的溶解度: ≥ 1 mg/mL (3.20 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 10.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 2 中的溶解度: 10 mg/mL (32.01 mM) in 50% PEG300 50% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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