AMPK/mTOR/ULK1 signaling pathway [2]

GK pretreatment (50 μM, 24 h) promoted astrocyte proliferation after oxygen-glucose deprivation (OGD) as shown by increased cell viability (CCK-8 assay) and increased number of EdU-positive cells compared to OGD alone [2].

GK pretreatment enhanced astrocyte migration after OGD, evidenced by increased number of migrated cells (Transwell assay) and increased migration distance (wound scratch assay) [2].

GK pretreatment upregulated autophagy-related protein 7 (ATG7), Beclin-1, and the LC3-II/LC3-I ratio, while downregulating p62 protein expression in astrocytes after OGD, indicating induced autophagy [2].

GK pretreatment increased phosphorylated AMPK (p-AMPK) and phosphorylated ULK1 (p-ULK1) levels, and decreased phosphorylated mTOR (p-mTOR) levels in astrocytes following OGD, demonstrating activation of the AMPK/mTOR/ULK1 signaling pathway [2].

Co-treatment with Compound C (10 μM, an AMPK inhibitor) reversed GK-induced upregulation of p-AMPK and p-ULK1, downregulation of p-mTOR, and blocked GK-induced autophagy flux (reversed ATG7, LC3-II, Beclin-1, and p62 changes); moreover, Compound C abolished GK’s promotory effects on astrocyte proliferation and migration after OGD [2].

Primary astrocytes were isolated from the cortical hemispheres of 2-day-old rats. Cells were cultured in glial medium with 5% horse serum at 37°C in a 5% CO2 humidified incubator until 90% confluence, then oligodendrocytes and microglia were removed by shaking at 180 rpm overnight. To mimic ischemia/reperfusion, astrocytes were washed three times in serum- and glucose-free glial medium, then incubated in an anaerobic chamber (94% N2, 5% CO2, 1% O2) for 3 h at 37°C. After OGD, cells were reoxygenated in normal glial medium under normoxic conditions (95% air, 5% CO2) for indicated times (6, 12, or 24 h) [2].

For drug treatment, astrocytes were pretreated with GK (50 μM) for 24 h before OGD. In some experiments, the AMPK inhibitor Compound C (10 μM) was added 24 h before OGD together with GK. Cell viability was assessed at 24, 48, 72, 96 h post-OGD using CCK-8 assay: 10 μL of CCK-8 reagent was added to each well, incubated for 1 h at 37°C, and optical density at 450 nm was measured [2].

Cell proliferation was measured by EdU assay: astrocytes were cultured in medium containing 50 μM EdU for 2 h, fixed in methanol for 15 min, treated with 0.5% Triton X-100 for 20 min, stained with Apollo® reaction cocktail for 30 min and Hoechst33342 for 30 min, then EdU-positive cells were visualized and counted [2].

Migration was evaluated by Transwell chamber assay: migration inserts were coated with fibronectin; 24 h after OGD, cells were resuspended in glial medium with 1% FBS and 100 μL of cell suspension (10^4 cells/mL) was seeded into the upper chamber, and migrated cells were counted [2].

Wound scratch assay: after OGD, astrocytes grown to 90% confluence were starved in serum-free glial medium for 24 h, then a scratch was made with a micropipette tip; residual cells were removed by PBS rinsing, and images were captured at 12 and 24 h post-incubation to measure migration distance [2].

Western blot analysis: total protein was extracted with ice-cold RIPA buffer; 30 μg of total protein was separated by 15% SDS-PAGE and transferred to PVDF membranes; membranes were blocked with 5% skim milk and probed with primary antibodies against ATG7, LC3, Beclin-1, p62, AMPK, p-AMPK (Thr172), mTOR, p-mTOR (Ser2448), ULK1, p-ULK1 (Ser757), GFAP, and β-actin, followed by HRP-conjugated secondary antibody; signal was detected and band intensity was analyzed [2].

[1]. Ginkgolide K promotes astrocyte proliferation and migration after oxygen-glucose deprivation via inducing protective autophagy through the AMPK/mTOR/ULK1 signaling pathway. Eur J Pharmacol. 2018 Aug 5;832:96-103.

Ginkgolide K (GK) is a derivative compound of ginkgolide B extracted from the leaves of Ginkgo biloba. It has been used to treat ischemic stroke due to its neuroprotective potential. In this study, GK promoted astrocyte proliferation and migration after oxygen-glucose deprivation and reoxygenation via inducing protective autophagy through the AMPK/mTOR/ULK1 signaling pathway, suggesting GK might be a potential agent for cerebral ischemia/reperfusion injury [2].

C20H22O9

406.39

406.126

153355-70-5

101553595

White to off-white solid powder

1.6±0.1 g/cm3

743.5±60.0 °C at 760 mmHg

269.8±26.4 °C

0.0±5.6 mmHg at 25°C

1.649

0.48

2

9

1

29

946

9

CC1=C2[C@H]([C@@H]([C@@]34[C@@]25C(=O)O[C@@H]3C[C@H]([C@@]46[C@H](C(=O)O[C@H]6O5)O)C(C)(C)C)O)OC1=O

MGXAKXRVQRODDX-GNQXGQJISA-N

InChI=1S/C20H22O9/c1-6-9-10(27-13(6)23)11(21)19-8-5-7(17(2,3)4)18(19)12(22)14(24)28-16(18)29-20(9,19)15(25)26-8/h7-8,10-12,16,21-22H,5H2,1-4H3/t7-,8+,10+,11-,12-,16-,18-,19-,20-/m0/s1

(1R,3R,6R,7S,8S,10R,11R,12R,13R)-8-tert-Butyl-6,12-dihydroxy-16-methyl-2,4,14,19-tetraoxahexacyclo[8.7.2.01,11.03,7.07,11.013,17]nonadec-16-ene-5,15,18-trione

GKGinkgolide K

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

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

DMSO : ~50 mg/mL (~123.04 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.15 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.12 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 20.8 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 3: ≥ 2.08 mg/mL (5.12 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 20.8 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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 (Please use freshly prepared in vivo formulations for optimal results.)