Epilepsy is a common chronic neurological disorder worldwide, but its entire pathology remains unknown. The purpose of this study was to explore the antiepileptic effect of baicalin (BAL), the main bioactive component of scutellaria.
We isolated astrocytes from neonatal rats and astrocytes were identified by glial fibrillary acidic protein (GFAP) immunostaining. The viability and phenotype of astrocytes were determined by Cell Counting Kit-8 (CCK-8) and immunofluorescence staining, respectively.
For investigating the effect of BAL on the autophagy in A1 astrocytes treated PC12 cells, expression of light chain 3B (LC3-B) and sequestosome 1 (P62) was analyzed by immunofluorescence staining and apoptosis by acridine orange/ethidium bromide (AO/EB) staining, respectively.
For animal experiments, pentylenetetrazol (PTZ)-induced epileptic model was used to explore the antiepileptic effect of BAL. The results showed that BAL reduced lipopolysaccharide (LPS)-induced complement C3 (C3, a marker of A1 astrocytes)+ A1 cells and decreased autophagy and apoptosis in PC12 cells. Further findings showed seizure grade and latency were positively correlated with GFAP+/C3+ A1 cells’ infiltration in interstitial astrocytes. After BAL treatment, epileptogenesis was ameliorated with decreased A1 astrocytes in the brain and improved behavioral performance.
The enzyme-linked immunosorbent assay (ELISA) showed that the levels of interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α) were reduced in the cerebral interstitial site in the BAL group compared to the PTZ group.
Western blotting analysis showed that BAL treatment reduced expression of C3, inward rectifier potassium channel Kir4.1, aquaporin-4 (AQP4) in the frontal cortex and Caspase-3, BCL2-associated X protein (Bax) in the hippocampus. In conclusion, these findings suggest that BAL can prevents cognitive and emotional disorders and has antiepileptic effects in rats, which may be associated with suppresses neuron autophagy and apoptosis in the hippocampus via regulate astrocyte phenotypes.
Protective Effects of Chaga Medicinal Mushroom, Inonotus obliquus (Agaricomycetes), Extract on β-Amyloid-Induced Neurotoxicity in PC12 Cells and Aging Rats: In Vitro and In Vivo Studies
Modulation of β-amyloid (Aβ)-induced neurotoxicity has emerged as a possible therapeutic approach to ameliorate the onset and progression of Alzheimer’s disease. The present study aimed to evaluate the protective effect of Inonotus obliquus extracts (IOEs) on Aβ-induced neurotoxicity in cultured rat pheochromocytoma (PC12) cells and rats.
Exposure of PC12 cells to IOE significantly elevated cell viability, decreased intracellular calcium levels, and attenuated Aβ-mediated cell apoptosis. In aging rats, IOE can decrease the production of amyloid precursor protein (APP) and the levels of Aβ plaques in hippocampus after IOE treatment in the brain by an action that is associated with a lowering of the of IL-1β, TNF-α levels.
Our findings indicate that IOE has potential neuroprotective actions against Aβ-induced neurotoxicity, which may occur through modulation of calcium channels or downstream molecules involved in inflammation.
Membrane bound catechol-O-methytransferase is the dominant isoform for dopamine metabolism in PC12 cells and rat brain
Impaired dopamine activity in the dorsolateral prefrontal cortex (DLPFC) is thought to contribute to cognitive deficits in diseases such as schizophrenia, attention deficit hyperactivity disorder (ADHD) and traumatic brain injury. Catechol-O-methyltransfease (COMT) metabolizes dopamine and is an important regulator of dopamine signaling in the DLPFC.
In mammalian species, two isoforms of COMT protein, membrane-bound COMT (MB-COMT) and soluble COMT (S-COMT), are encoded by one COMT gene and expressed widely.
While S-COMT is thought to play a dominant role in the peripheral tissues, MB-COMT is suggested to have a greater role in dopamine metabolism in the brain. However, whether a selective inhibitor for MB-COMT may effectively block dopamine metabolism remains unknown.
We generated a knockout of MB-COMT in PC12 cells using CRISPR-cas9 technology to evaluate the effect of both MB and S-COMT on dopamine metabolism.
Deletion of MB-COMT in PC12 cells significantly decreased homovanillic acid (HVA), completely depleted 3-methyoxytyramine (3-MT), and significantly increased 3,4-dihydroxyphenylacetic acid (DOPAC) levels. Comparison of the effect of a MB-COMT selective inhibitor LI-1141 on dopamine metabolism in wild type and MB-COMT knockout PC12 cells allowed us to confirm the selectivity of LI-1141 with respect to MB-COMT in cells.
Under conditions in which LI-1141 was shown to inhibit only MB-COMT but not S-COMT, it effectively changed dopamine metabolites similar to the effect induced by tolcapone, a non-selective COMT inhibitor, suggesting that selective inhibition of MB-COMT will be effective in blocking dopamine metabolism, providing an attractive therapeutic approach in improving cognition for patients.
Bisphenol A and rotenone induce S-nitrosylation of protein disulfide isomerase (PDI) and inhibit neurite outgrowth of primary cultured cells of the rat hippocampus and PC12 cells
Bisphenol A (BPA) interferes the function and development of the central nervous system (CNS), resulting in behavioral abnormalities and memory loss. S-nitrosylation of protein disulfide isomerase (PDI) is increased in brains with sporadic Alzheimer’s disease and Parkinson’s disease.
The aim of the present study was to clarify the role of nitric oxide (NO) in BPA-induced neurotoxicity. Since rotenone induces NO-mediated neurodegeneration through S-nitrosylation of PDI, it was used as a positive control. First, rats were treated with BPA and rotenone, and S-nitrosylation of PDI was detected in rat brain microsomes.
BPA and rotenone decreased RNase oxidation activity of PDI concomitant with S-nitrosylation of PDI. Next, to clarify S-nitrosylation of PDI by BPA and rotenone in rat brains, we treated the rat pheochromocytoma cell line PC12 and primary cultured neuron cells from the rat hippocampus with BPA (5 and 10 μM) and rotenone (100 or 200 nM).
BPA induced S-nitrosylation of PDI, while NG-monomethyl-L-arginine (L-NMMA), a NOS inhibitor, exerted the opposite effects. Finally, to evaluate the toxicity of BPA in the CNS, we investigated its effects on neurite outgrowth of PC12 and primary cultured neuron cells.
BPA inhibited neurite outgrowth of these cells, while L-NMMA reversed this inhibition. The involvement of PDI activity in neurite outgrowth was also examined, and bacitracin, a PDI inhibitor, is shown to decrease neurite outgrowth.
Furthermore, the overexpression of PDI, but not a catalytically inactive PDI mutant, enhanced neurite outgrowth. These results suggested that S-nitrosylation of PDI induced by excessive NO caused BPA-induced neurotoxicity.
Characterizing ischaemic tolerance in rat pheochromocytoma (PC12) cells and primary rat neurons
Preconditioning tissue with sublethal ischaemia or hypoxia can confer tolerance (protection) against subsequent ischaemic challenge. In vitro ischaemic preconditioning (IPC) is typically achieved through oxygen glucose deprivation (OGD), whereas hypoxic preconditioning (HPC) involves oxygen deprivation (OD) alone.
Here, we report the effects of preconditioning of OGD, OD or glucose deprivation (GD) in ischaemic tolerance models with PC12 cells and primary rat neurons. PC12 cells preconditioned (4 h) with GD or OGD, but not OD, prior to reperfusion (24 h) then ischaemic challenge (OGD 6 h), showed greater mitochondrial activity, reduced cytotoxicity and decreased apoptosis, compared to sham preconditioned PC12 cells.
Furthermore, 4 h preconditioning with reduced glucose (0.565 g/L, reduced from 4.5 g/L) conferred protective effects, but not for higher concentrations (1.125 or 2.25 g/L). Preconditioning (4 h) with OGD, but not OD or GD, induced stabilization of hypoxia inducible factor 1α (HIF1α) and upregulation of HIF1 downstream genes (Vegf, Glut1, Pfkfb3 and Ldha). In primary rat neurons, only OGD preconditioning (4 h) conferred neuroprotection.
OGD preconditioning (4 h) induced stabilization of HIF1α and upregulation of HIF1 downstream genes (Vegf, Phd2 and Bnip3). In conclusion, OGD preconditioning (4 h) followed by 24 h reperfusion induced ischaemic tolerance (against OGD, 6 h) in both PC12 cells and primary rat neurons.
The OGD preconditioning protection is associated with HIF1α stabilization and upregulation of HIF1 downstream gene expression. GD preconditioning (4 h) leads to protection in PC12 cells, but not in neurons.
This GD preconditioning-induced protection was not associated with HIF1α stabilization.