Glucocorticoid Receptor

Glucocorticoids remain the most widely used immunosuppressive and anti-inflammatory drugs, yet substantial gaps exist in our understanding of glucocorticoid-mediated immunoregulation. To address this, we generated a pathway-level map of the transcriptional effects of glucocorticoids on nine primary human cell types. This analysis revealed that the response to glucocorticoids is highly cell type dependent, in terms of the individual genes and pathways affected, as well as the magnitude and direction of transcriptional regulation. Based on these data and given their importance in autoimmunity, we conducted functional studies with B cells. We found that glucocorticoids impair upstream B cell receptor and Toll-like receptor 7 signaling, reduce transcriptional output from the three immunoglobulin loci, and promote significant up-regulation of the genes encoding the immunomodulatory cytokine IL-10 and the terminal-differentiation factor BLIMP-1. These findings provide new mechanistic understanding of glucocorticoid action and emphasize the multifactorial, cell-specific effects of these drugs, with potential implications for designing more selective immunoregulatory therapies[2].

Methylprednisolone acetate (30 mg/kg, intramuscular injection; additional oral dose of 13 mg/kg, for 10 consecutive days) combined with LPS induces the typical characteristics of early AVN of the femoral head [2].

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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].

Bone marrow-derived mononuclear cells were collected by incubation in hemolysis solution. Mononuclear cells were fixed in ethanol (70%) for 24 hours and stained with propidium iodide for 10 minutes. Cell cycle analysis was done by flow cytometry[2].

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Choice of glucocorticoid and glucocorticoid concentration [2]Methylprednisolone was based on two facts. First, this glucocorticoid is commonly used in clinical practice when rapid immunosuppression is required. Second, it has dose-linear pharmacokinetics, which makes it easier to estimate plasma concentrations from the doses that are commonly used in the clinic (Möllmann et al., 1989; Derendorf et al., 1991). For the initial in vitro studies, in which cells were treated with methylprednisolone and the transcriptional response was studied by RNA-seq, we chose to treat the cells in vitro with a concentration of 22.7 µM, which was estimated to be equivalent to the peak plasma concentration after an intravenous dose of 1 g (a dose commonly used in acute presentations of autoimmune diseases). When we performed the in vivo studies of glucocorticoid response in circulating human B cells, the study volunteers were given a dose of 250 mg. We measured methylprednisolone levels in the plasma of each volunteer, and the mean value of the methylprednisolone concentration at 4 h was 5.34 µM, which is very close to what we had predicted based on previous data (Möllmann et al., 1989; Derendorf et al., 1991) and on the dose-linear pharmacokinetics of the drug. To bring the in vitro conditions as close as possible to those of our in vitro study, all subsequent experiments used a methylprednisolone concentration of 5.34 µM.

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In vitro glucocorticoid treatment for RNA-seq [2]Methylprednisolone 22.7 µM (Sigma; cat. no. M0639) was added to two of the wells and vehicle (ethanol, 0.08%) to the other two. At 2 and 6 h after the stimulus, cells were harvested from one of the wells that received vehicle and one of the wells that received methylprednisolone. Immediately after harvesting, the cells originating from each well were independently separated from the supernatant by centrifugation, resuspended in 500 μl of TRIzol Reagent (Thermo Fisher Scientific; cat. no. 15596018), and stored at −80°C until the time of RNA purification. All downstream processing steps (RNA purification, RNA-seq library preparation, and sequencing) were performed separately for each well.

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Differential expression analysis for RNA-seq data [2]Methylprednisolone-treated versus vehicle-treated cells in each of four subjects, it was also important to choose a method that allows a paired analysis. Methylprednisolone-treated versus vehicle-treated cells at each time point, with vehicle-treated cells as the reference, r takes the values 0 or 1: Xj0 = 1, Xj1 = 0 for the vehicle-treated cells and Xj0 = 0, Xj1 = 1 for the methylprednisolone-treated cells. The log fold change in gene i is reflected by βi1 − βi1. To improve efficiency in assessing the effect of methylprednisolone treatment, where gene expression is measured in cells from the same subjects treated with methylprednisolone or vehicle, we performed a paired analysis by adding a subject effect into the above negative binomial model, following the method of Love et al. (2014).

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

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)

Solubility in Formulation 1: ≥ 1.67 mg/mL (4.01 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 1.67 mg/mL (4.01 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.67 mg/mL (4.01 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)