Endogenous Metabolite; gama-aminobutyric acid (GABA)

An extremely potent PAF antagonist cage molecule called Ginkgolide A (BN-52020) was extracted from the leaves of the Ginkgo biloba tree. shows promise in a broad range of immune and inflammatory disorders. Ginkgolide A (BN-52020) did not reduce apoptotic damage in neurons treated with staurosporine or those devoid of serum[2].

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Impact of GA/Ginkgolide A on chondrocyte viability To assess the impact of varying concentrations of Ginkgolide A/GA and different incubation times on chondrocyte viability, we employed the CCK-8 assay. After 24 h of incubation, GA concentrations below 50 μmol exhibited minimal cytotoxicity to chondrocytes (Fig. 2B). Additionally, at a 50 μmol concentration, GA had negligible effects on chondrocyte viability over 24 h of incubation (Fig. 2C). To simulate oxidative stress in chondrocytes, 30 μmol of TBHP was used [25], and chondrocyte viability was assessed at 12, 24, and 48 h with varying GA concentrations (Fig. 2D). Results indicated that GA at 50 μmol for 24 h provided optimal protective effects.

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A inhibits ERS, chondrocyte apoptosis, and ECM degradation [4]
Building on GO functional enrichment results, the mechanisms by which GA/Ginkgolide A protects chondrocytes were further evaluated. Toluidine Blue staining was used to assess ECM levels. TBHP treatment significantly reduced Toluidine Blue staining in chondrocytes, an effect countered by GA, leading to more intense purple staining (Fig. 2E). Similar results were seen in type II collagen immunofluorescence staining, where GA reversed TBHP-induced Collagen II suppression (Fig. 2F, G). Immunoblotting revealed that GA significantly mitigated TBHP-induced ECM degradation, as indicated by the reduced expression of MMP13 and ADAMTS5 and increased expression of Collagen II (Fig. 3A-D). These findings suggest that GA exerts a protective effect on the ECM. Subsequently, ROS levels were measured using DCFH-DA fluorescence probes, demonstrating that GA significantly reduced ROS levels elevated by TBHP (Fig. 3E, F). TUNEL staining confirmed that TBHP increased chondrocyte apoptosis, whereas GA pre-treatment provided substantial protection (Fig. 3G, H). Protein analysis revealed that TBHP treatment elevated Cleaved-Caspase 3 and BAX expression while reducing BCL-2 levels (Fig. 3I-L). Western blotting indicated that ERS-related proteins, including p-PERK, GRP78, ATF4, p-eIF2α, and CHOP, were significantly reduced as GA concentration increased (Fig. 3M-R). These findings confirm that GA inhibits oxidative stress-induced ERS, chondrocyte apoptosis, and ECM degradation.

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GA/Ginkgolide A inhibits ERS to reduce chondrocyte apoptosis and ECM degradation [4]
Building on earlier findings that Ginkgolide A/GA protects chondrocytes, we investigated whether this involves ERS mitigation. To test this hypothesis, thapsigargin (TG) was used to specifically induce ERS. Flow cytometry was used to assess apoptosis levels post-TG treatment, revealing significantly increased late apoptosis (Annexin V-FITC+/PI+) compared to the GA+TBHP group (Fig. 4A, B). Immunoblotting showed that TG treatment reduced Collagen II levels while increasing CHOP, GRP78, and Cleaved-Caspase-3 levels compared to the GA+TBHP group (Fig. 4C-G). Similarly, immunofluorescence staining of the ERS-related protein GRP78 indicated that TG augmented ERS activity (Fig. 4H, I). These results suggest that TG counteracts GA's protective effects on chondrocytes. Our data indicate that GA inhibits oxidative stress-induced ERS, thereby reducing chondrocyte apoptosis and ECM degradation.

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GA/Ginkgolide A upregulates FoxO1 expression [4]
To further explore the upstream pathways through which Ginkgolide A/GA may regulate ERS, KEGG pathway enrichment analysis was conducted based on shared targets between GA and OA. Results suggested that activation of the FoxO pathway might be a critical factor in GA's mitigation of oxidative stress (Fig. 5C). Molecular docking was used to analyze the interaction between GA and FoxO1. Semi-flexible docking indicated significant binding activity, with binding energy distributions being mainly below −5 kcal/mol (Fig. 5A). PyMOL analysis identified optimal molecular conformations where GA formed hydrogen bonds with Leu239 and Lys179 residues of FoxO1, suggesting these residues are key for the substrate binding and catalytic activity (Fig. 5B). Molecular docking demonstrated strong binding energy between GA and FoxO1. It was hypothesized that GA upregulates FoxO1 expression. Western blot and immunofluorescence analyses confirmed that TBHP treatment suppressed FoxO1 expression in chondrocytes, while GA significantly enhanced FoxO1 expression in a dose-dependent manner (Fig. 5D-F). Immunofluorescence further corroborated that GA increased FoxO1 expression and reduced ERS protein CHOP in TBHP-treated chondrocytes (Fig. 5G, H). Based on our results, GA was found to upregulate FoxO1 expression.

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GA/Ginkgolide A inhibits ERS to reduce chondrocyte apoptosis and ECM degradation by upregulating FoxO1 [4]
To investigate whether FoxO1 silencing could reverse Ginkgolide A/GA's anti-apoptotic and anti-ECM degradation effects, Western blotting was conducted. Silencing FoxO1 reversed GA's upregulation of FoxO1 (Fig. 6A-C). Immunofluorescence confirmed that FoxO1 silencing negated GA-mediated upregulation of FoxO1 and downregulation of CHOP in TBHP-treated chondrocytes (Fig. 6D-F). Additionally, FoxO1 silencing significantly increased ERS-related proteins, including p-PERK, GRP78, ATF4, p-eIF2α, and CHOP, in GA-treated chondrocytes (Fig. 6G-L). Additionally, Silencing FoxO1 eliminated GA's anti-apoptotic and anti-ECM degradation effects. Western blot analysis indicated that GA treatment reduced MMP13, Cleaved-Caspase 3, ADAMTS5, and BAX expression, while FoxO1 silencing increased these markers (Fig. 6M, O, P, S). GA-induced upregulation of Collagen II and BCL-2 in TBHP-treated chondrocytes was reversed by FoxO1 silencing (Fig. 6N, R). These findings confirm that GA regulates chondrocyte apoptosis and ECM degradation by enhancing FoxO1 expression and inhibiting ERS.

In sedated mice, ginkgolide A dramatically reduced their amount of time spent sleeping[1].

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The aim of this study is to investigate whether Ginkgolide A (GA) can improve TBI in mice and whether it can alleviate cell apoptosis in the brain of TBI mice by reducing oxidative stress. Mice received TBI and GA administration for 7 days. Neurological deficits were monitored and brain tissues were examined for molecular pathological markers. TBI mice had more severer neurobehavioral deficits compared with sham group, which could be improved by administration of GA. GA administration improveed Modified Neurological Severity Scale (mNSS) scores, Grid-Walking test and Rotarod test of TBI mice. The apoptosis increased in TBI mice, and reduced after GA treatment. The biomarkers of oxidative stress 8-OHdG and malondialdehyde (MDA) in the brain of TBI mice increased, while SOD reduced. These changes were reversed after GA administration. These outcomes showed that GA could raise neurobehavioral deficiency of TBI mice. GA treatment could attenuate apoptosis in TBI mice by reducing oxidative stress.[3]

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This study aimed to investigate the effects of Ginkgolide A (GA) on chondrocytes under oxidative stress and to elucidate its potential molecular mechanisms. Using a destabilization of the medial meniscus (DMM) model in mice and an in vitro osteoarthritis (OA) model induced by tert-butyl hydroperoxide (TBHP) in chondrocytes, we validated the therapeutic efficacy and underlying mechanisms of GA. Potential OA targets of GA were identified through network pharmacology, Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Further exploration into the effects on endoplasmic reticulum stress (ERS), apoptosis, extracellular matrix (ECM) degradation, and Forkhead Box O1 (FoxO1) related pathways was conducted using Western blotting, immunofluorescence, TUNEL staining, flow cytometry, X-ray, micro-computed tomography (Micro-CT) analysis, and histological staining. The results demonstrated that GA upregulated FoxO1 expression and inhibited ERS-related signaling pathways, thereby reducing apoptosis and ECM degradation. In conclusion, GA significantly alleviated OA symptoms both in vitro and in vivo, suggesting its potential as a therapeutic agent for OA [4].

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GA/Ginkgolide A activates FoxO1 in vivo and ameliorates OA progression post-DMM surgery [4]
In vivo studies investigated the therapeutic effects of GA/Ginkgolide A on OA, with different concentrations administered intraperitoneally to DMM mouse models. The drug safety of GA was confirmed through HE staining of key organs in each group (Fig. S1A). X-ray analysis, Micro-CT analysis, HE, SO, and Toluidine Blue staining assessed histopathological changes in joint cartilage. X-rays revealed significant cartilage sclerosis and joint space narrowing in the DMM group compared to sham-operated controls, while GA treatment showed partial improvement in OA progression (Fig. 7A). Further analysis using Micro-CT revealed a significant increase in the bone volume to tissue volume (BV/TV) ratio of the subchondral bone in the DMM group. However, GA treatment resulted in a marked, concentration-dependent reduction in the BV/TV ratio (Fig. 7B, D). Cartilage sections stained with HE, SO, and Toluidine Blue showed pronounced cartilage damage and extensive proteoglycan loss in the DMM group, which was alleviated by GA treatment (Fig. 7C). The severity of OA in each experimental group was assessed using the OARSI and synovial scoring systems, both of which were evaluated independently by two researchers under blinded conditions, which showed the highest scores in the DMM group, and a dose-dependent reversal of OA symptoms in the GA-treated groups (Fig. 7E,F). Immunohistochemical analysis of tissue samples from all mice indicated reduced FoxO1 and Collagen II expression and increased CHOP and Cleaved-Caspase 3 in the OA group (Fig. 7G–K). Notably, GA application effectively reversed these changes. Collectively, GA demonstrated significant efficacy in mitigating OA progression in the DMM mouse model.

Cell viability assay [4]
We utilized the CCK-8 assay to evaluate the viability of mouse chondrocytes. Following established protocols, approximately 8,000 cells were seeded per well in a 96-well plate. After 24 h of incubation, drug treatments (e.g. Ginkgolide A) were applied to determine the optimal concentrations and incubating time. Cell viability was ultimately measured by optical density at 450 nm using a microplate reader.

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Identification of GA/Ginkgolide A targets and hypothetical OA-related proteins [4]
In our research, we used the PharmMapper database (https://www.lilab-ecust.cn/pharmmapper/) to identify potential protein targets of Ginkgolide A/GA, focusing particularly on human protein targets. Concurrently, we gathered information on target genes associated with OA from the GeneCards database (https://www.genecards.org/). The protein–protein interaction (PPI) network for the intersecting targets was constructed using Cytoscape (version 3.10.0).

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Molecular docking [4]
In this study, to explore the molecular interaction between GA/Ginkgolide A and the FoxO1 protein, we obtained the crystal structure of the FoxO1 (PDB ID: 5DUI) from the RCSB PDB database (https://www.rcsb.org/). We conducted 200 semi-flexible docking experiments using AutoDock software (version 4.2) to obtain the binding energy distribution and schematic diagrams of the optimal molecular binding sites. The optimal conformation exhibited a binding affinity of −6.09 kcal/mol. In the optimal conformation, Ginkgolide A/GA formed hydrogen bonds with the Leu239 and Lys179 residues of FoxO1.

TBI mouse model and GA/Ginkgolide A treatment [3]
Induced TBI by a controlled cortical impact device. Anesthetized the mice with isoflurane (1.5–2.5 %). Drilled a 5 mm diameter hole on the right cerebral hemisphere (2.0 mm posterior from bregma and 2.0 mm lateral to the sagittal suture) after retracting the scalp and fascia to expose the dura. Used a 3 mm-flat impactor tip to impact the exposed dual (impact parameters: velocity: 3.0 m/s, depth: 2 mm, dwell time: 100 ms). After the surgery, sutured the scalp incision, and placed the mice in a heating pad until anesthesia was restored. Sham-injured mice underwent all these procedures without any impact. Simultaneously, injected Ginkgolide A/GA intraperitoneally once a day for 7 days (5 mg/kg), and physiological saline was used as the carrier. Lesion volume was assessed in accordance with previous reports

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Superoxide dismutase (SOD) activity level [3]
Seven days after GA/Ginkgolide A administration, the mice were sacrificed under deep anesthesia. The brain tissue was quickly removed from the ice dish, and the damaged brain tissue was selected. Used a microplate reader to measure superoxide dismutase (SOD) according to the manufacturer's instructions.

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Immunofluorescence [3]
Fixed the brain samples with 4 % paraformaldehyde, embedded in paraffin, and cut into 5-μm-thick slides. Then, incubated the samples with primary antibody against Bax, CC3 and 8-hydroxy-2' -deoxyguanosine (8-OHdG) at 4 °C overnight. Then, incubated them with corresponding secondary antibodies at room temperature for 2 h. After that, performed nuclear cells re-staining with 4’,6-diamidino-2-phenylindole. Captured the images with a fluorescence microscope. Five fields were randomly chosen from each section of the three to five consecutive brain sections from each rat.

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This study utilized 24 male C57BL/6 mice, aged 8 weeks. A mouse model of OA was established using DMM surgery. After a two-week acclimation period, the mice were divided into four groups: sham surgery control, DMM model, DMM with low-dose GA/Ginkgolide A (20 mg/kg), and DMM with high-dose GA/Ginkgolide A (40 mg/kg). Eight weeks post-surgery, the mice were euthanized, and knee joint tissues were harvested for histological examination.[4]

[1]. Isolation of bilobalide and ginkgolide A from Ginkgo biloba L. shorten the sleeping time induced in mice by anesthetics. Biol Pharm Bull, 1993. 16(2): p. 210-2.

[2]. Pharmacological studies supporting the therapeutic use of Ginkgo biloba extract for Alzheimer's disease. Pharmacopsychiatry, 2003. 36(S 1): p. 8-14.

[3]. Ginkgolide A attenuated apoptosis via inhibition of oxidative stress in mice with traumatic brain injury. Heliyon. 2024 Jan 14;10(2):e24759.

[4]. Ginkgolide A enhances FoxO1 expression and reduces endoplasmic reticulum stress to mitigate osteoarthritis in mice. Int Immunopharmacol. 2024 Dec 5;142(Pt B):113116.

Ginkgolide A is a highly active PAF antagonist cage molecule that is isolated from the leaves of the Ginkgo biloba tree. Shows potential in a wide variety of inflammatory and immunological disorders.
ginkgolide-A has been reported in Machilus wangchiana and Ginkgo biloba with data available.
See also: Ginkgo (part of).

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The leaves of Ginkgo biloba L. and aqueous extract from them shortened the sleeping time induced in mice by anesthetics (hexobarbital, alpha-chloralose and urethane, i.p.). Two characteristic terpenoids in G. biloba, bilobalide and Ginkgolide A, significantly shortened the sleeping time induced by anesthetics. A toxic substance, 4-O-methylpyridoxine (MPN), responsible for "gin-nan food poisoning" isolated from the seed of G. biloba, was not detected from the extract of the leaves of G. biloba. Therefore, the Ginkgo biloba extract has no toxicities for MPN.[1]

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There are two limitations in this present study. Firstly, we found GA/Ginkgolide A administration significantly alleviated the apoptosis in the brain of TBI mice. However, the apoptotic pathways are still unclear. Previous reviews have summarized ginkgolides alleviated many related apoptosis pathway, as evidenced by decreasing p-JNK, p-PERK, p-IRE1α, ATF6, C/EBP homologous protein, toll-like receptor 4/nuclear factor kappa-B, and the PI3K/Akt pathways in several diseases. In the future, we will explore which pathways are involved in the regulation of GA on the apoptosis in the brain of TBI mice. Secondly, endoplasmic reticulum stress modulator such as docosahexaenoic acid (DHA), or additional oxidative stress modulators such as lipoic acid can be as therapeutic for prevention of chronic traumatic encephalopathy. We will further investigate how GA can be combined with DHA or lipoic acid.
Conclusion: We found that GA/Ginkgolide A treatment could improve neurological functions of TBI mice. Administration of GA alleviated neurological function by reducing cell apoptosis and oxidative stress of TBI mice. GA could be a drug for TBI therapy in the future.[3]

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In summary, GA/Ginkgolide A enhances FoxO1 expression in chondrocytes, protecting them from the activation of the PERK-ATF4-eIF2α-CHOP pathway while mitigating apoptosis and ECM degradation. Our study primarily explores the interactions between FoxO1 and the PERK-ATF4-eIF2α-CHOP network in GA-treated chondrocytes. As a key transcription factor, FoxO1 activity is regulated by various mechanisms, including acetylation, phosphorylation, ubiquitination, and complex formation with other transcription factors. Although our study did not elucidate the specific mechanisms by which GA regulates FoxO1 expression, it demonstrated that GA reduces chondrocyte apoptosis and TBHP-induced ERS-mediated ECM degradation in mouse chondrocytes, thereby protecting joints from OA-related damage. These effects are achieved through FoxO1 upregulation and inhibition of the PERK-ATF4-eIF2α-CHOP pathway. Similarly, the relationship between FoxO1 and endoplasmic reticulum stress is complex and multifaceted. Previous studies have demonstrated that the activation of FoxO1 can induce autophagy, thereby facilitating the degradation of misfolded proteins and damaged organelles, which in turn alleviates the impact of endoplasmic reticulum stress. However, in this study, it remains uncertain whether FoxO1 influences the stress response pathway solely through the regulation of PERK, or if it concurrently affects multiple protein targets. Additionally, whether FoxO1 directly regulates PERK by binding to its protein domains or indirectly modulates PERK through post-translational modifications such as ubiquitination or phosphorylation, remains unresolved. These questions necessitate further investigation in future studies. Considering clinical application challenges, we used intraperitoneal injection in animal models, yielding favorable results. Different GA administration routes, such as oral, subcutaneous, and intra-articular injections, warrant further exploration. Future research should focus on other potential signaling pathways and clinically appropriate GA delivery methods. Another limitation of this study is the absence of a positive control, both in vivo and in vitro, to compare the effects of GA. The schematic diagram of our findings is shown in Fig. 8. Despite certain limitations, our preliminary results provide new directions for effective OA management and treatment .[4]

C20H24O9

408.4

408.142

C, 58.82; H, 5.92; O, 35.26

15291-75-5

9909368

White to off-white solid powder

1.6±0.1 g/cm3

710.1±60.0 °C at 760 mmHg

280°C (dec.)

256.5±26.4 °C

0.0±5.1 mmHg at 25°C

1.631

-0.13

2

9

1

29

893

10

C[C@@H]1C(=O)O[C@@H]2[C@]1([C@@]34C(=O)O[C@H]5[C@]3(C2)[C@@]6([C@@H](C5)C(C)(C)C)[C@H](C(=O)O[C@H]6O4)O)O

FPUXKXIZEIDQKW-VKMVSBOZSA-N

InChI=1S/C20H24O9/c1-7-12(22)26-10-6-17-9-5-8(16(2,3)4)18(17)11(21)13(23)28-15(18)29-20(17,14(24)27-9)19(7,10)25/h7-11,15,21,25H,5-6H2,1-4H3/t7-,8+,9-,10+,11+,15+,17-,18+,19-,20-/m1/s1

9H-1,7a-(Epoxymethano)-1H,6aH-cyclopenta(c)furo(2,3-b)furo(3,2:3,4)cyclopenta(1,2-d)furan-5,9,12(4H)-trione, 3-(1,1-dimethylethyl)hexahydro-4,7b-dihydroxy-8-methyl-, (1R,3S,3aS,4R,6aR,7aR,7bR,8S,10aS,11aS)-

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.12 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 25.0 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: ≥ 2.5 mg/mL (6.12 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 25.0 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: ≥ 2.5 mg/mL (6.12 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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 (Please use freshly prepared in vivo formulations for optimal results.)