Glucocorticoid Receptor

糖皮质激素仍然是应用最广泛的免疫抑制和抗炎药，但我们对糖皮质激素介导的免疫调节的理解存在很大差距。为了解决这一问题，我们生成了糖皮质激素对九种主要人类细胞类型的转录作用的通路水平图。这项分析表明，就受影响的单个基因和途径以及转录调控的大小和方向而言，对糖皮质激素的反应是高度依赖于细胞类型的。基于这些数据并考虑到它们在自身免疫中的重要性，我们对B细胞进行了功能研究。我们发现糖皮质激素损害上游B细胞受体和Toll样受体7信号传导，降低三个免疫球蛋白基因座的转录输出，并促进编码免疫调节细胞因子IL-10和末端分化因子BLIMP-1的基因的显著上调。这些发现为糖皮质激素作用提供了新的机制理解，并强调了这些药物的多因素、细胞特异性作用，对设计更具选择性的免疫调节疗法具有潜在意义[2]。

醋酸甲泼尼龙（30 mg/kg，肌内注射；追加口服剂量13 mg/kg，连续10天）联合LPS可诱导股骨头早期AVN的典型特征[2]。

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成年小鼠随机分为实验组和对照组。A组（实验组）通过肌肉注射给予10mg/kg脂多糖（LPS）和30mg/kg甲基泼尼松龙（MPS）。每只小鼠还连续10天分次口服13 mg/kg的多磺酸粘多糖。B组（对照组）在与A组相同的位置和相同的体积接受生理盐水。在最后一次化学注射后3、5和7周，通过电子显微镜观察股骨头的组织学变化。随机测量空腔隙的百分比，并使用图像分析系统评估纤维软骨的表达。通过免疫组织化学观察CD31和VEGF-R2的表达。骨髓来源的单核细胞用碘化丙啶染色，并通过流式细胞术分析细胞周期[2]。

通过在溶血溶液中孵育收集骨髓来源的单核细胞。将单核细胞在乙醇（70%）中固定24小时，并用碘化丙啶染色10分钟。通过流式细胞术进行细胞周期分析[2]。

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糖皮质激素和糖皮质激素浓度的选择[2]Methylprednisolone的选择基于两个事实。首先，当需要快速免疫抑制时，这种糖皮质激素通常用于临床实践。其次，它具有剂量线性药代动力学，这使得更容易根据临床常用的剂量估算血浆浓度（Möllmann等人，1989；Derendorf等人，1991）。对于最初的体外研究，其中细胞用甲基泼尼松龙处理，并通过RNA-seq研究转录反应，我们选择以22.7µM的浓度在体外处理细胞，估计相当于静脉注射1g剂量（通常用于自身免疫性疾病急性发作的剂量）后的峰值血浆浓度。当我们对循环人类B细胞中的糖皮质激素反应进行体内研究时，研究志愿者服用了250mg的剂量。我们测量了每位志愿者血浆中的甲基泼尼松龙水平，4小时时甲基泼尼松浓度的平均值为5.34µM，这与我们根据之前的数据（Möllmann等人，1989；Derendorf等人，1991）和药物的剂量线性药代动力学预测的非常接近。为了使体外条件尽可能接近我们的体外研究，所有后续实验都使用5.34µM的甲基泼尼松龙浓度。

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糖皮质激素对RNA-seq的体外治疗[2]甲基泼尼松龙22.7µM（Sigma；目录号M0639）加入其中两个孔中，并将赋形剂（乙醇，0.08%）加入另外两个孔。在刺激后2小时和6小时，从其中一个接受载体的孔和一个接受甲基泼尼松龙的孔中收获细胞。收获后，立即通过离心将来自每个孔的细胞与上清液独立分离，重新悬浮在500μl TRIzol试剂（赛默飞世尔科技公司；目录号15596018）中，并在-80°C下储存，直至RNA纯化。对每个孔分别进行所有下游处理步骤（RNA纯化、RNA-seq文库制备和测序）。

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RNA-seq数据的差异表达分析[2]甲基泼尼松龙治疗的细胞与载体治疗的细胞的数据，选择一种允许配对分析的方法也很重要。 甲基泼尼松龙处理的细胞与溶媒处理的细胞，r取0或1的值：赋形剂处理的电池Xj0=1，Xj1=0，甲基泼尼松处理的电池Xj0=0，Xj1=1。基因i的对数倍数变化由βi1−βi1反映。为了提高评估甲基泼尼松龙治疗效果的效率，我们按照Love等人（2014）的方法，通过将受试者效应添加到上述负二项模型中，进行了配对分析。

Animal/Disease Models: Femoral necrosis mouse model methylprednisolone and lipose-induced head [2]
Doses: 30 mg/kg; 13 mg/kg for 10 days
Route of Administration: 30 mg/kg, intramuscularinjection ;Additional oral dose of 13 mg/kg for 10 days resulted in chondrocyte degeneration and fibrocartilage expression after 7 weeks. The density of CD31 and VEGF-R2 markers increased in the femoral head.

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Adult mice were randomly divided into two groups: experimental and control. Group A (the experimental group) was given (via intramuscular injection) 10 mg/kg of lipopolysaccharide (LPS) and 30 mg/kg of methylprednisolone (MPS). Each mouse additionally received MPS in divided oral doses of 13 mg/kg for 10 consecutive days. Group B (the control group) received normal saline at the same location and same volume as those in Group A. Histological changes of the femoral heads were observed by electron microscopy at 3, 5, and 7 weeks after the last chemical injection. The percentage of empty lacunae was measured randomly and the expression of fibrocartilage was evaluated using an image analyzing system. The expression of CD31 and VEGF-R2 were observed by immunohistochemistry. The bone marrow-derived mononuclear cells were stained with propidium iodide and cell cycle was analyzed by flow cytometry.[2]

[1]. Immune regulation by glucocorticoids can be linked to cell type-dependent transcriptional responses. J Exp Med. 2019 Feb 4;216(2):384-406.

[2]. A mouse model of osteonecrotic femoral head induced by methylprednisolone and liposaccharide. Biomedical Research and Therapy volume 3, Article number: 12 (2016).

Methylprednisolone acetate is an acetate ester resulting from the formal condensation of the 21-hydroxy function of 6alpha-methylprednisolone compound with acetic acid. It has a role as an anti-inflammatory drug. It is a 20-oxo steroid, a 17alpha-hydroxy steroid, an 11beta-hydroxy steroid, a glucocorticoid, an acetate ester, a steroid ester, a 3-oxo-Delta(1),Delta(4)-steroid and a tertiary alpha-hydroxy ketone. It is functionally related to a 6alpha-methylprednisolone.
Methylprednisolone Acetate is the acetate salt of a synthetic glucocorticoid receptor agonist with immunosuppressive and antiinflammatory effects. Methylprednisolone acetate is converted into active prednisolone in the body, which activates glucocorticoid receptor mediated gene expression. This includes inducing synthesis of anti-inflammatory protein IkappaB-alpha and inhibiting synthesis of nuclear factor kappaB (NF-kappaB). As a result, proinflammatory cytokine production such as IL-1, IL-2 and IL-6 is down-regulated and cytotoxic T-lymphocyte activation is inhibited. Therefore, an overall reduction in chronic inflammation and autoimmune reactions may be achieved.
Methylprednisolone derivative that is used as an anti-inflammatory agent for the treatment of ALLERGY and ALLERGIC RHINITIS; ASTHMA; and BURSITIS; and for the treatment of ADRENAL INSUFFICIENCY.
See also: Methylprednisolone (has active moiety); Methylprednisolone acetate; neomycin sulfate (component of).

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Introduction: Osteonecrosis of the femoral head is caused by various factors, including prolonged use of steroid drugs, use of alcohol, vascular injuries and hemoglobinopathies. This study aims to develop a mouse model for glucocorticoid-induced avascular necrosis (AVN) of the femoral head.
Methods: Adult mice were randomly divided into two groups: experimental and control. Group A (the experimental group) was given (via intramuscular injection) 10 mg/kg of lipopolysaccharide (LPS) and 30 mg/kg of methylprednisolone (MPS). Each mouse additionally received MPS in divided oral doses of 13 mg/kg for 10 consecutive days. Group B (the control group) received normal saline at the same location and same volume as those in Group A. Histological changes of the femoral heads were observed by electron microscopy at 3, 5, and 7 weeks after the last chemical injection. The percentage of empty lacunae was measured randomly and the expression of fibrocartilage was evaluated using an image analyzing system. The expression of CD31 and VEGF-R2 were observed by immunohistochemistry. The bone marrow-derived mononuclear cells were stained with propidium iodide and cell cycle was analyzed by flow cytometry.
Results: The results showed that at weeks 3 and 5, mice in Group A showed an increase in body weight. From weeks 5 to 7, mouse body weight in both groups remained constant. No difference in bone morphology was observed at week 7. The percentage of empty lacunae was 5.87 ± 2.49% at week 5 and 21.58 ± 8.10% at week 7. After 7 weeks, chondrocyte degeneration and fibrocartilage expression were observed. Moreover, the density of CD31 and VEGF-R2 markers increased in the femoral head. The rate of apoptosis in the bone marrow increased at week 3 then decreased.
Conclusion: The data show that MPS, combined with LPS, can induce in mice features typical of early AVN of the femoral head.[2]

C24H32O6

416.50728

416.219

C, 69.21; H, 7.74; O, 23.05

53-36-1

Methylprednisolone;83-43-2

5877

Typically exists as white to off-white solids at room temperature

1.3±0.1 g/cm3

582.5±50.0 °C at 760 mmHg

206ºC

196.5±23.6 °C

0.0±3.7 mmHg at 25°C

1.580

3.08

100.9

2

6

4

30

858

8

CC(OCC(C1(CCC2C3CC(C)C4=CC(C=CC4(C)C3C(CC12C)O)=O)O)=O)=O

PLBHSZGDDKCEHR-LFYFAGGJSA-N

InChI=1S/C24H32O6/c1-13-9-16-17-6-8-24(29,20(28)12-30-14(2)25)23(17,4)11-19(27)21(16)22(3)7-5-15(26)10-18(13)22/h5,7,10,13,16-17,19,21,27,29H,6,8-9,11-12H2,1-4H3/t13-,16-,17-,19-,21+,22-,23-,24-/m0/s1

2-((6S,8S,9S,10R,11S,13S,14S,17R)-11,17-dihydroxy-6,10,13-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl acetate

Lemod; Methylprednisolone acetate; Depo M-Predrol; 15847-24-2; (11beta,20R)-11,17,20,21-Tetrahydroxypregna-1,4-dien-3-one; DTXSID30553372; DTXCID60504155; 20(R)-Hydroxy Prednisolone; (8S,9S,10R,11S,13S,14S,17R)-17-[(1R)-1,2-dihydroxyethyl]-11,17-dihydroxy-10,13-dimethyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthren-3-one; (20R)-11beta,17alpha,20,21-Tetrahydroxypregna-1,4-dien-3-one; (20R)-Hydroxyprednisolone; Depo-Medrate Depo-medrol;Depo-Medrin Depomedrone; Depometicort Medrol Methyl prednisolone acetate; Methylprednisolone 21-acetate; NSC 48985; Medrol acetate; Mepred

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 : ≥ 100 mg/mL (~240.09 mM)

配方 1 中的溶解度: ≥ 1.67 mg/mL (4.01 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% 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 16.7 mg/mL澄清的DMSO储备液加入到400 μL PEG300中，混匀；再向上述溶液中加入50 μL Tween-80，混匀；然后加入450 μL生理盐水定容至1 mL。
*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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配方 2 中的溶解度: ≥ 1.67 mg/mL (4.01 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 16.7mg/mL澄清的DMSO储备液加入到900μL 20％SBE-β-CD生理盐水中，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 3 中的溶解度: ≥ 1.67 mg/mL (4.01 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (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 16.7 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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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));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
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；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。