Rat Pheochromocytoma PC12 Cells Research Articles

Anti-oxidative stress and cognitive improvement of a semi-synthetic isoorientin-based GSK-3β inhibitor in rat pheochromocytoma cell PC12 and scopolamine-induced AD model mice via AKT/GSK-3β/Nrf2 pathway

BackgroundAlzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive deficits. Although the pathogenesis of AD is unclear, oxidative stress has been implicated to play a dominant role in its development. The flavonoid isoorientin (ISO) and its synthetic derivatives TFGF-18 selectively inhibit glycogen synthase kinase-3β (GSK-3β), a potential target of AD treatment. PurposeTo investigate the neuroprotective effect of TFGF-18 against oxidative stress via the GSK-3β pathway in hydrogen peroxide (H2O2)-induced rat pheochromocytoma PC12 cells in vitro and scopolamine (SCOP)-induced AD mice in vivo. MethodThe oxidative stress of PC12 cells was induced by H2O2 (600 μM) and the effects of TFGF-18 (2 and 8 μM) or ISO (12.5 and 50 μM) were observed. The AD mouse model was induced by SCOP (3 mg/kg), and the effects of TFGF-18 (2 and 8 mg/kg), ISO (50 mg/kg), and donepezil (DNP) (3 mg/kg) were observed. DNP, a currently accepted drug for AD was used as a positive control. The neuronal cell damages were analyzed by flow cytometry, LDH assay, JC-1 assay and Nissl staining. The oxidative stress was evaluated by the detection of MDA, SOD, GPx and ROS. The level of ACh, and the activity of AChE, ChAT were detected by the assay kit. The expressions of Bax, Bcl-2, caspase3, cleaved-caspase3, p-AKT (Thr308), AKT, p-GSK-3β (Ser9), GSK-3β, Nrf2, and HO-1, as well as p-CREB (Ser133), CREB, and BDNF were analyzed by western blotting. Morris water maze test was performed to analyze learning and memory ability. ResultsTFGF-18 inhibited neuronal damage and the expressions of Bax caspase3 and cleaved-caspase3, and increased the expression of Bcl-2 in vitro and in vivo. The level of MDA and ROS were decreased while the activities of SOD and GPx were increased by TFGF-18. Moreover, TFGF-18 increased the p-AKT, p-GSK-3β (Ser9), Nrf2, HO-1, p-CREB, and BDNF expression reduced by H2O2 and SCOP. Meanwhile, MK2206, an AKT inhibitor, reversed the effect of TFGF-18 on the AKT/GSK-3β pathway. In addition, the cholinergic system (ACh， ChAT, and AChE) disorders were retrained and the learning and memory impairments were prevented by TFGF-18 in SCOP-induced AD mice. ConclusionsTFGF-18 protects against neuronal cell damage and cognitive impairment by inhibiting oxidative stress via AKT/GSK-3β/Nrf2 pathway.

Adrenal G protein-coupled Receptor Kinase (GRK)-2 Downregulation And Regulator of G protein Signaling (RGS)-4 Upregulation Mediate The Sympatholytic Effect Of Dapagliflozin

Dapagliflozin is a sodium/­glucose co-­transporter (SGLT)-­2 inhibitor with beneficial cardiovascular effects independent of its anti­diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering, beneficial in chronic human heart failure (HF). Adrenal G protein­-coupled receptor kinase (GRK)-­2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity in HF due to dysregulation of adrenal sympatho-­inhibitory α2­-adrenergic receptors (ARs). On the other hand, we recently reported that Regulator of G protein Signaling (RGS)-­4 inhibits free fatty acid receptor (FFAR)-­3-stimulated NE release from sympathetic neurons, while it is also known to inhibit adrenal CA secretion induced by acetylcholine. Thus, we hypothesized herein that dapagliflozin may lower SNS hyperactivity by enhancing adrenal α2AR and RGS4 functions. We used the rat pheochromocytoma PC12 cell line expressing human α2A­AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217±67 pg/ml, n=6), compared to vehicle-­treated rats (363±77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-­treated vs. vehicle­-treated adrenal glands. In contrast, RGS4 mRNA and protein levels were increased by dapagliflozin ­treatment. Adrenal α2AR density was higher in dapagliflozin- ­vs. vehicle-­treated rats (51.3±7.3 vs. 26.1±8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e., GRK2 & TH downregulation with RGS4 upregulation) were completely recapitulated in PC12 cells in culture, treated with 5 μM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR signaling towards inhibition of CA secretion was markedly enhanced, while the cholinergic agonist carbachol-induced CA secretion was reduced post-­dapagliflozin treatment of PC12 cells. GRK2 overexpression and RGS4 siRNA-mediated knockdown partially reversed dapagliflozin‘s effect on CA secretion, confirming the involvement of GRK2 and RGS4 in dapagliflozin‘s sympatholytic effects in PC12 cells. In conclusion, dapagliflozin exerts adrenal sympatholysis via: a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization (i.e., enhances α2AR sympatho-inhibitory signaling); and b) upregulation of RGS4, which directly opposes cholinergic-stimulated CA secretion. 1) NIH/NHLBI #HL155718-01. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Metformin inhibits nerve growth factor-induced sympathetic neuron differentiation through p35/CDK5 inhibition.

The authors' previous research has shown the pivotal roles of cyclin-dependent kinase 5 (CDK5) and its regulatory protein p35 in nerve growth factor (NGF)-induced differentiation of sympathetic neurons in PC12 cells. During the process of differentiation, neurons are susceptible to environmental influences, including the effects of drugs. Metformin is commonly used in the treatment of diabetes and its associated symptoms, particularly in diabetic neuropathy, which is characterized by dysregulation of the sympathetic neurons. However, the impacts of metformin on sympathetic neuronal differentiation remain unknown. In this study, we investigated the impact of metformin on NGF-induced sympathetic neuronal differentiation using rat pheochromocytoma PC12 cells as a model. We examined the regulation of TrkA-p35/CDK5 signaling in NGF-induced PC12 differentiation. Our results demonstrate that metformin reduces NGF-induced PC12 differentiation by inactivating the TrkA receptor, subsequently inhibiting ERK and EGR1. Inhibition of this cascade ultimately leads to the downregulation of p35/CDK5 in PC12 cells. Furthermore, metformin inhibits the activation of the presynaptic protein Synapsin-I, a substrate of CDK5, in PC12 differentiation. In addition, metformin alters axonal and synaptic bouton formation by inhibiting p35 at both the axons and axon terminals in fully differentiated PC12 cells. In summary, our study elucidates that metformin inhibits sympathetic neuronal differentiation in PC12 cells by disrupting TrkA/ERK/EGR1 and p35/CDK5 signaling. This research contributes to uncovering a novel signaling mechanism in drug response during sympathetic neuronal differentiation, enhancing our understanding of the intricate molecular processes governing this critical aspect of neurodevelopment.NEW & NOTEWORTHY This study unveils a novel mechanism influenced by metformin during sympathetic neuronal differentiation. By elucidating its inhibitory effects from the nerve growth factor (NGF) receptor, TrkA, to the p35/CDK5 signaling pathways, we advance our understanding of metformin's mechanisms of action and emphasize its potential significance in the context of drug responses during sympathetic neuronal differentiation.

Abstract 1971: Olverembatinib, a novel multikinase inhibitor, demonstrates superior antitumor activity in succinate dehydrogenase (SDH)-deficient neoplasms

Abstract SDH deficiency, characterized by loss of SDHB, occurs in many tumors including paraganglioma, pheochromocytoma, renal, and gastrointestinal stromal tumor (GIST). Olverembatinib, a novel multikinase inhibitor, targets a broad spectrum of kinases and has promising efficacy in SDH-deficient GIST (Qiu H et al. J Clin Oncol. 2023). Here, we assessed antitumor effects of olverembatinib in preclinical models of SDH-deficient cancers and explored potential mechanisms of action (MOA). Antiproliferation assays were conducted using CellTiter-Glo®. MOA analyses were performed using western blotting. In vivo activity was evaluated using an SDHB knock-out [KO])-derived mouse xenograft model. Olverembatinib had superior antiproliferative activity (vs other approved TKIs) in SDH-deficient cell lines (IC50, 0.129-5.132 μM), including Jurkat clone E6-1 (SDHB mutation [mut]), OS-RC-2 (SDHA and SDHB mut), RKO (SDHA mut), and rat pheochromocytoma PC12 cells with SDHB KO (PC12#5F7) (Table 1). In PC12#5F7 cells, olverembatinib decreased HIF-1α, HIF-2α, VEGFA, and FGFR1 protein levels and inhibited phosphorylation of FGFR1, IGF-1R, SRC, AKT, and ERK1/2. Increased cleavage of caspase-3 and PARP-1 was observed, suggesting induction of apoptosis. In PC12#5F7 (SDHB KO)-derived xenograft models, olverembatinib demonstrated dose-dependent antitumor activity at 10 and 20 mg/kg (QOD), with tumor growth inhibition rates of 27.4% and 52.2%, respectively. At 20 mg/kg, olverembatinib showed superior efficacy vs ponatinib (10 mg/kg) and rogaratinib (50 mg/kg). Results from western blot analyses were similar in both tumor tissues and cell lines. Olverembatinib exerts promising antitumor effects in SDH-deficient neoplasms and can modulate multiple signal pathways involved in angiogenesis, apoptosis, proliferation, and survival. The results provide a preclinical rationale for future development of olverembatinib in SDH-deficient cancers. Table 1. Antiproliferation activity of TKIs in SDH-deficient cell lines IC50 μM, mean ± SD Cell lines Acute lymphoblastic leukemia Renal cell carcinoma Colon cancer Rat pheochromocytoma carcinoma Jurkat, clone E6-1 OS-RC-2 RKO PC12 PC12#5F7 SDH status* SDHB: missense_variant, p.A15T SDHA: missense_variant, p.G184R; SDHB: stop gained, p.W218Ter SDHA: missense_variant, p.P279S SDHB competent SDHB exon 2+3 KO (heterozygote) by CRISPR-Cas9 Compound Olverembatinib (KIT, PDGFR, SRC, VEGFR, FGFR, FLT-3, RET) 0.149 ± 0.04 0.135 ± 0.083 0.129 ± 0.136 9.600 ± 1.407(n = 4) 5.132 ± 1.950 Imatinib (KIT, PDGFR) > 10 > 10 > 10 > 10 > 10 Ripretinib (KIT, PDGFR) 1.559 ± 0.294 8.781 ± 1.725 1.247 ± 0.933 > 10 > 10 Avapritinib (PDGFRA exon 18 mut) > 8.071 ± 2.728 > 10 ± 0 > 10 ± 0 > 10 > 10 Ponatinib (KIT, PDGFR, VEGFR, FGFR) 2.136 ± 2.107 0.809 ± 0.443 0.274 ± 0.241 > 10 8.537 ± 3.553 Dasatinib (KIT, SRC, PDGFR, FGFR) 2.025 ± 0.644 0.029 ± 0.003 0.382 ± 0.320 > 10 > 10 Sunitinib (VEGFR) 4.289 ± 0.280 > 5.099 ± 0.207 3.789 ± 0.385 > 10 > 10 Regorafenib (VEGFR) > 10 5.110 ± 0.588 2.145 ± 0.812 > 10 > 10 Sorafenib (VEGFR) > 10 3.581 ± 0.255 3.875 ± 0.170 > 10 > 10 Pazopanib (VEGFR, FGFR) > 10 > 10 > 10 > 10 > 10 Rogaratinib (FGFR) > 10 > 10 > 10 > 10 > 10 Infigratinib (FGFR) 4.581 ± 0.342 3.166 ± 0.791 2.374 ± 2.311 > 10 > 10 Pemigatinib (FGFR) 4.642 ± 1.842 > 10 > 10 > 10 > 10 *From CrownBio database. Citation Format: Yan Xiong, Eric Liang, Ping Min, Huidan Yu, Bingxing Wu, Guoqin Zhai, Dajun Yang, Yifan Zhai. Olverembatinib, a novel multikinase inhibitor, demonstrates superior antitumor activity in succinate dehydrogenase (SDH)-deficient neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1971.

Hybrid pharmacophore design and synthesis of donepezil-inspired aurone derivative salts as multifunctional acetylcholinesterase inhibitors

Flavonoids, a ubiquitous group of plant polyphenols, are well-known for their beneficial effects on human health. Their phenylchromane skeletons have structural similarities to donepezil [the US FDA-approved drug used to treat Alzheimer's disease (AD)]. The objective of this study was to design and synthesize valuable agents derived from flavonoids for relieving the symptoms of AD. A variety of flavonoid derivative salts incorporating benzylpyridinium units were synthesized and several of them remarkedly inhibited acetylcholinesterase (AChE) activity in vitro. Additionally, aurone derivative salts protected against cell death resulting from t-BHP exposure in rat pheochromocytoma PC12 cells and slightly promoted neurite outgrowth. Furthermore, they potently suppressed the aggregation of amyloid-β (Aβ1-42). Our findings highlight the effectiveness of donepezil-inspired aurone derivative salts as multipotent candidates for AD.

MicroRNA-194-5p/Heparin-binding EGF-like growth factor signaling mediates dexamethasone-induced activation of pseudorabies virus in rat pheochromocytoma cells

Pseudorabies virus (PRV) is a neurotropic virus, which infects a wide range of mammals. The activity of PRV is gradually suppressed in hosts that have tolerated the primary infection. Increased glucocorticoid levels resulting from stressful stimuli overcome repression of PRV activity. However, the host cell mechanism involved in the activation processes under stressful conditions remains unclear. In this study, infection of rat PC-12 pheochromocytoma cells with neuronal properties using PRV at a multiplicity of infection (MOI) = 1 for 24 h made the activity of PRV be the relatively repressed state, and then incubation with 0.5 μM of the corticosteroid dexamethasone (DEX) for 4 h overcomes the relative repression of PRV activity. RNA-seq deep sequencing and bioinformatics analyses revealed different microRNA and mRNA profiles of PC-12 cells with/without PRV and/or DEX treatment. qRT-PCR and western blot analyses confirmed the negative regulatory relationship of miRNA-194–5p and its target heparin-binding EGF-like growth factor (Hbegf); a dual-luciferase reporter assay revealed that Hbegf is directly targeted by miRNA-194–5p. Further, miRNA-194–5p mock transfection contributed to PRV activation, Hbegf was downregulated in DEX-treated PRV infection cells, and Hbegf overexpression contributed to returning activated PRV to the repression state. Moreover, miRNA-194–5p overexpression resulted in reduced levels of HBEGF, c-JUN, and p-EGFR, whereas Hbegf overexpression suppressed the reduction caused by miRNA-194–5p overexpression. Overall, this study is the first to report that changes in the miR-194–5p-HBEGF/EGFR pathway in neurons are involved in DEX-induced activation of PRV, laying a foundation for the clinical prevention of stress-induced PRV activation.

Dapagliflozin downregulates G protein-coupled receptor Kinase-2 and upregulates regulator of g protein signaling-4 in adrenals to exert sympatholysis in heart failure

Abstract Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor with beneficial cardiovascular effects independent of its anti-diabetic actions. Among these is sympatholysis, i.e., norepinephrine (NE) lowering which is beneficial in chronic human heart failure (HF). Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity in HF due to dysregulation of adrenal sympatho-inhibitory α2-adrenergic receptors (ARs) [1]. On the other hand, we recently reported that signaling by neuronal free fatty acid receptor (FFAR)-3, which stimulates NE release from sympathetic neurons, is blocked by Regulator of G protein Signaling (RGS)-4 [2,3]. We hypothesized herein that dapagliflozin may lower SNS hyperactivity in the adrenal gland by improving α2AR, and antagonizing FFAR3 signaling in adrenal chromaffin cells. We used the rat pheochromocytoma PC12 cell line expressing human α2A-AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p<.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands. In contrast, RGS4 mRNA levels were increased in dapagliflozin-treated adrenals. Adrenal α2AR density was higher in dapagliflozin- vs. vehicle-treated rats (51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p<.05). These results (i.e., GRK2 & TH downregulation with RGS4 upregulation) were completely recapitulated in PC12 cells in culture, treated with 5 microM dapagliflozin (or vehicle) for 24 hours. Importantly, α2AR-dependent G protein-mediated signaling towards inhibition of CA secretion was markedly enhanced, whereas FFAR3-dependent phospholipase C/calcium signaling was reduced, at 24 hrs post-dapagliflozin treatment of PC12 cells. GRK2 overexpression partially reversed dapagliflozin`s effect on cholinergic-induced CA secretion. In conclusion, dapagliflozin exerts a sympatholytic action in the adrenal medulla via a) downregulation of TH, which reduces CA biosynthesis, and GRK2, which reduces α2AR desensitization; and b) upregulation of RGS4 to block FFAR3-stimulated CA secretion (Figure).Figure

Bone Marrow Mesenchymal Stem Cell Exosomal miR-345-3p Ameliorates Cerebral Ischemia-reperfusion Injury by Targeting TRAF6.

The purpose of this study was to investigate the effects of bone marrow mesenchymal stem cells (BMSCs) exosomal miR-345-3p and tumor necrosis factor receptorassociated factor 6 (TRAF6) on cerebral ischemia reperfusion (CIR) injury. Exosomes (Exos) derived from BMSCs were isolated and identified. PC12 (rat pheochromocytoma) cells were used to establish an oxygen and glucose deprivation/reoxygenation (OGD/R) model. Cell counting kit-8, TUNEL staining, lactate dehydrogenase staining, RT-qPCR, and western blotting were utilized for analyzing the functions of miR-345-3p about PC12 cells. Dualluciferase reporter experiment was then to confirm the link between miR-345-3p and TRAF6. Finally, using male SD rats, the middle cerebral artery occlusion (MCAO) model was constructed. Regulation of I/R damage in MCAO rats of miR-345-3p and TRAF6 were further explored in the changes of modified neurological severity score, cerebral infarction pictures, relative infarct volume, and histopathological changes. After OGD/R treatment, neuronal apoptosis was dramatically increased. After treatment with exosomal miR-345-3p, OGD/R-induced neuroapoptosis was dramatically inhibited. Exosomal miR-345-3p inhibited OGD/R-induced neuroapoptosis by downregulating the expression of TRAF6. However, the miR-345-3p inhibitor aggravated the changes caused by OGD/R. The corresponding regulations of miR-345-3p were reversed with TRAF6 overexpression. The animal experiments in vivo further verified that miR-345-3p ameliorated brain I/R injury in MCAO rats by targeting TRAF6. This study found that BMSCs-exosomal miR-345-3p protected against CIR injury by decreasing TRAF6.

Chemical constituents of Wikstroemia alternifolia and their neuroprotective activities

Phytochemical investigation on the plant of Wikstroemia alternifolia led to the isolation of 26 compounds including two new ones, wikstralternifols A and B (1 and 7). Their structures including the absolute configuration were elucidated by spectroscopic data together with analysis of experimental and calculated ECD data. All compounds were isolated from this plant for the first time, and their main structural types were lignans, sesquiterpenoids, and flavonoids. In the sodium nitroprusside-induced rat pheochromocytoma PC-12 cell model, the neuroprotective activities of the selected sesquiterpenoids (1 and 4) and lignans (7 − 14) were screened at the concentration of 10 µM, and 7 − 14 displayed better activities than the positive control edaravone.

Prenylated dihydroflavones from the root barks of Morus alba

Three new prenylated dihydroflavones, moralbaflavones A–C (1–3), together with four known ones (4a/4b, 5, and 6) were isolated from the root barks of Morus alba L. Their structures including the absolute configurations were determined by the analysis of HRMS, NMR, and ECD data. The neuroprotective properties of these prenylated dihydroflavones were screened at the concentration of 10 µM in the sodium nitroprusside-induced rat pheochromocytoma PC-12 cells, and the results showed moralbaflavone C (3) possessed significant neuroprotective activity, being more potent than the positive control edaravone.

Snake venom nerve growth factor-inspired designing of novel peptide therapeutics for the prevention of paraquat-induced apoptosis, neurodegeneration, and alteration of metabolic pathway genes in the rat pheochromocytoma PC-12cell.

Neurodegenerative disorders (ND), associated with the progressive loss of neurons, oxidative stress-mediated production of reactive oxygen species (ROS), and mitochondrial dysfunction, can be treated with synthetic peptides possessing innate neurotrophic effects and neuroprotective activity. Computational analysis of two small synthetic peptides (trideca-neuropeptide, TNP; heptadeca-neuropeptide, HNP) developed from the nerve growth factors from snake venoms predicted their significant interaction with the human TrkA receptor (TrkA). In silico results were validated by an in vitro binding study of the FITC-conjugated custom peptides to rat pheochromocytoma PC-12cell TrkA receptors. Pre-treatment of PC-12cells with TNP and HNP induced neuritogenesis and significantly reduced the paraquat (PT)-induced cellular toxicity, the release of lactate dehydrogenase from the cell cytoplasm, production of intracellular ROS, restored the level of antioxidants, prevented alteration of mitochondrial transmembrane potential (ΔΨm) and adenosine triphosphate (ATP) production, and inhibited cellular apoptosis. These peptides lack in vitro cytotoxicity, haemolytic activity, and platelet-modulating properties and do not interfere with the blood coagulation system. Functional proteomic analyses demonstrated the reversal of PT-induced upregulated and downregulated metabolic pathway genes in PC-12cells that were pre-treated with HNP and revealed the metabolic pathways regulated by HNP to induce neuritogenesis and confer protection against PT-induced neuronal damage in PC-12. The quantitative RT-PCR analysis confirmed that the PT-induced increased and decreased expression of critical pro-apoptotic and anti-apoptotic genes had been restored in the PC-12cells pre-treated with the custom peptides. A network gene expression profile was proposed to elucidate the molecular interactions among the regulatory proteins for HNP to salvage the PT-induced damage. Taken together, our results show how the peptides can rescue PT-induced oxidative stress, mitochondrial dysfunction, and cellular death and suggest new opportunities for developing neuroprotective drugs.

Fast and Sustained Axonal Growth by BDNF Released from Chitosan Microspheres.

Brain-derived neurotrophic factor (BDNF) regulates dendritic branching and dendritic spine morphology, as well as synaptic plasticity and long-term potentiation. Consequently, BDNF deficiency has been associated with some neurological disorders such as Alzheimer's, Parkinson's or Huntington's diseases. In contrast, elevated BDNF levels correlate with recovery after traumatic central nervous system (CNS) injuries. The utility of BDNF as a therapeutic agent is limited by its short half-life in a pathological microenvironment and its low efficacy caused by unwanted consumption of non-neuronal cells or inappropriate dosing. Here, we tested the activity of chitosan microsphere-encapsulated BDNF to prevent clearance and prolong the efficacy of this neurotrophin. Neuritic growth activity of BDNF release from chitosan microspheres was observed in the PC12 rat pheochromocytoma cell line, which is dependent on neurotrophins to differentiate via the neurotrophin receptor (NTR). We obtained a rapid and sustained increase in neuritic out-growth of cells treated with BDNF-loaded chitosan microspheres over control cells (p < 0.001). The average of neuritic out-growth velocity was three times higher in the BDNF-loaded chitosan microspheres than in the free BDNF. We conclude that the slow release of BDNF from chitosan microspheres enhances signaling through NTR and promotes axonal growth in neurons, which could constitute an important therapeutic agent in neurodegenerative diseases and CNS lesions.

Effect of Withania Somnifera (L.) root extract\'s on PC12 induced by hydrogen peroxide, study

The L. is also used as a crude medicament that mostly effected in various neurological and immunological disorders. It is also known as Ashwagandha and “Indian ginseng” in Indian region. There is a lack of information on the possible neuroprotective properties of W. somnifera root against HO- and Ab(1-42)-induced cytotoxicity, which are now targeted for innovative treatments for dementia, particularly dementia of the Alzheimer's type (AD). According to this research, we prepared an aqueous extract of dried roots of W. somnifera that possess the protective effect against Ab- aggregated fibril and HO cytotoxicity through MTT assay with the help of differentiated rat pheochromocytoma PC12 cell. The findings indicate that, in a concentration-dependent manner, pretreatment of differentiated PC12 cells with aqueous extracts of W. somnifera root strongly protects differentiated PC12 cells against both H2O2- and Ab(1-42)-induced cytotoxicity. The W. somnifera extract was examined using liquid chromatography-serial mass spectrometry in order to look into the substances that could be responsible for the effects that were seen. Withanolide derivatives, notably withaferin A, were found in abundance. These findings support the hypothesis that W. somnifera may be used ethnopharmacologically to treat oxidative stress-related cognitive and other neurodegenerative illnesses. They also show the neuroprotective activities of an aqueous extract of W. somnifera root.

Biologically active drimane derivatives isolated from submerged cultures of the wood-inhabiting basidiomycete Dentipellis fragilis.

Four previously undescribed drimane sesquiterpenoids were isolated from submerged cultures of the wood-inhabiting basidiomycete Dentipellis fragilis along with two compounds that were previously reported as synthetic or biotransformation compounds but not as natural products. The constitution and relative configuration of these compounds was determined based on high-resolution electrospray ionization mass spectrometry as well as by 1D and 2D nuclear magnetic resonance spectroscopy. The absolute configurations were established based on exemplary calculation of circular dichroism spectra and comparison with measured data as well as on biogenetic considerations. The biological activities of the isolated compounds were assessed in antimicrobial, cytotoxicity and neurotrophic assays. 10-Methoxycarbonyl-10-norisodrimenin (3) exhibited weak activity against the Gram-positive bacterium Staphylococcus aureus and the zygomycete Mucor hiemalis with minimal inhibitory concentrations of 66.7 μg mL-1. In addition, compound 3 showed weak inhibition of the mammalian cell line KB3.1 (human endocervical adenocarcinoma) with a half maximal inhibitory concentration of 21.2 μM. The neurotrophic activities of 15-hydroxyisodrimenin (1) and 10-carboxy-10-norisodrimenin (5) were assed in neurite outgrowth and real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) assays. When supplemented with 5 ng mL-1 nerve growth factor (NGF), the drimanes 1 and 5 induced neurite outgrowth in PC-12 (rat pheochromocytoma) cells compared to cells solely treated with NGF. As evaluated by RT-qPCR, compounds 1 and 5 also increased NGF and brain-derived neurotrophic factor expression levels in 1321N1 astrocytoma cells. Interestingly, the current study only represents the second report on neurotrophic activities of this widespread class of terpenoids. The only other available study deals with Cyathus africanus, another basidiomycete that can produce drimanes and cyathanes, but is only distantly related to Dentipellis and the Hericiaceae.

Heterodimers with a cucurbitane-type triterpenoid skeleton from the branches of Elaeocarpus dubius

Four undescribed and two known cucurbitane-type triterpenoids, including two heterodimers, elaeocarpudubins A and B, were isolated from the branches of Elaeocarpus dubius (Elaeocarpaceae). The chemical structures of these undescribed isolates were determined by analyses of 1D and 2D NMR and MS data, electronic circular dichroism (ECD) calculations, and chemical transformation. Biogenetically, elaeocarpudubins A and B might be derived from cucurbitacin F through Michael addition with vitamin C and (−)-catechin, respectively. These six isolates were evaluated for their cytotoxic activities against human leukemia HL-60, human lung adenocarcinoma A549, human hepatoma SMMC-7721, human breast cancer MCF-7, human colon cancer SW480, and paclitaxel-resistant A549 (A549/Taxol) cell lines, for their antioxidant properties using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay, and for their differentiation effects on nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells. Cucurbitacins F (IC50 of 4.98–38.11 μM) and D (IC50 of 0.03–4.40 μM) showed growth-inhibitory activities against these six cancer cell lines. Elaeocarpudubin B (IC50 of 61.04 μM) and elaeocarpudoside B (IC50 of 6.93 μM) showed antioxidant activities. Elaeocarpudubin B and elaeocarpudoside B also showed neurite outgrowth-promoting activities in PC12 cells at a concentration of 10 μM.

New benzofuran neolignans with neuroprotective activity from Phyllanthodendron breynioides

A pair of undescribed dihydrobenzofuran neolignan enantiomers, (+/−)-phybrenan A (1a/1b), two new benzofuran neolignans, phybrenan B and C (2 and 3), along with four known neolignans (4 − 7) were obtained from the plants of Phyllanthodendron breynioides P. T. Li. The planar structures of all isolates were demonstrated by the analysis of detailed spectroscopic evidence (NMR, HRMS, and IR), and the absolute configurations of novel neolignans were elucidated by combined calculated and experimental ECD data analysis. The neuroprotective activities of all benzofuran neolignans against sodium nitroprusside (SNP)-induced cell death were examined in rat pheochromocytoma PC12 cells. The results exhibited that three compounds (4 − 6) possessed remarkable neuroprotective activities at 10 µM, better than the positive drug edaravone.

Abstract 11977: Dapagliflozin Downregulates Adrenal G Protein-Coupled Receptor-Kinase-2 to Exert Sympatholysis in Heart Failure

Introduction: Dapagliflozin is a sodium-glucose co-transporter (SGLT)-2 inhibitor exerting several cardiorenal protective effects independently of blood glucose regulation. Adrenal G protein-coupled receptor kinase (GRK)-2 upregulation is a major driver of circulating catecholamine (CA) elevation and sympathetic nervous system (SNS) hyperactivity, especially in heart failure (HF). GRK2 severely dysregulates adrenal sympatho-inhibitory α 2 -adrenergic receptors (ARs), leading to unchecked, chronically elevated CA secretion. Hypothesis: SGLT2 inhibition with dapagliflozin lowers SNS hyperactivity in part by opposing adrenal GRK2 actions. Methods: We used the rat pheochromocytoma PC12 cell line expressing human α 2A AR, as well as freshly isolated adrenal glands from rats treated with dapagliflozin in vivo. Results: Dapagliflozin treatment for 7 consecutive days (20 mg/kg/d in drinking water) led to a significant reduction in blood circulating NE levels (217+67 pg/ml, n=6), compared to control, vehicle-treated rats (363+77 pg/ml, n=6, p&lt;.05), suggesting reduced SNS activity. This was accompanied by reduced GRK2 and tyrosine hydroxylase (TH) mRNA and protein levels in dapagliflozin-treated rat adrenals vs. vehicle-treated animal-derived glands, indicating reduced adrenal CA synthesis and secretion. Finally, adrenal α 2 AR density was higher in dapagliflozin- vs. vehicle-treated rats(51.3+7.3 vs. 26.1+8.1 fmol/mg of protein, respectively; n=12 glands from 6 animals per group, p&lt;.05). GRK2 and TH downregulation were also observed in PC12 cells treated with 5 μM dapagliflozin for 24 hours. Importantly, α 2 AR-dependent inhibition of CA secretion was markedly enhanced 24 hrs post-dapagliflozin application. This was due to reduced GRK2-dependent receptor desensitization induced by dapagliflozin, since this effect was abolished in PC12 cells overexpressing GRK2. Conclusions: Dapagliflozin exerts a sympatholytic action in the adrenal medulla via downregulation of both TH, thus reducing CA biosynthesis, and GRK2, thus reducing α 2 AR desensitization to lower CA secretion. This adrenal-mediated sympatholysis may be part of the increasingly apparent cardiovascular benefits of SGLT2 inhibitors in human HF irrespective of diabetic status.

The Isolation and Preparation of Samwinol from Dracocephalum heterophyllum and Prevention on Aβ25-35-Induced Neuroinflammation in PC-12 Cells.

Dracocephalum heterophyllum (D. heterophyllum) is a traditional Chinese Tibetan medicine that has been used for the treatment of lymphitis, hepatitis, and bronchitis. However, only a few selected chemical components are currently obtained from D. heterophyllum, which limits its further pharmacological applications. In this study, we have obtained samwinol from D. heterophyllum by medium- and high-pressure liquid chromatography separation for the first time. Thereafter, we investigated the protective actions of samwinol against amyloid beta protein fragment 25–35 (Aβ25–35) induced neurotoxicity in cultured rat pheochromocytoma PC-12 cells and explored its underlying mechanisms of action. The results indicated that samwinol could increase cell viability and inhibit the production of reactive oxygen species (ROS) and mitochondria-derived ROS, as assessed by MTT assay, Giemsa staining, and flow cytometry assay. Through Western blot analysis, it was found that samwinol substantially inhibited the phosphorylation of ERK(1/2) and promoted the expression of HO-1 and Nrf2. The data obtained from molecular docking were also consistent with the above conclusions. All of these results showed that samwinol from D. heterophyllum can display significant anti-neuroinflammatory and antioxidant activities in vitro, which are associated with the suppression of ERK/AKT phosphorylation and the activation of the Nrf2/HO-1 signaling pathway. In the future, additional in-depth mechanism studies will be carried out to provide more evidence for the potential of samwinol in the treatment of Alzheimer’s disease.

Ribisins and Certain Analogues Exert Neuroprotective Effects through Activation of the Keap1-Nrf2-ARE Pathway.

Oxidative stress contributes to the pathogenesis of various neurodegenerative diseases and induction of the Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is a validated neuroprotective strategy. Synthetically-derived samples of members of the ribisin class of natural product together with a range of analogues were evaluated for their neuroprotective capacities. Four of the twenty-four compounds tested were found to strongly stimulate antioxidant response element-dependent transcriptional activity in human-derived SH-SY5Y cells. Further, in rat pheochromocytoma PC12 cells and mouse brain cortical cultures these compounds upregulated levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and its downstream target gene products, namely heme oxygenase (HO-1) and NAD(P)H quinone reductase 1 (NQO1). Functionally speaking, the compounds conferred protection in these cell models challenged with H2 O2 . In silico molecular modeling suggests that certain of the ribisins can dock in the Nrf2-binding Kelch domain in Keap1, while cysteine labeling by biotinylated iodoacetamide suggest that cysteine residues within Keap1 react with the ribisins. It is thus proposed that the most active compounds exert their neuroprotective activities by targeting Keap1, thereby activating Nrf2 and so increasing transactivation of Nrf2-responsive genes that encode for detoxifying and antioxidant enzymes.

Effects of Wnt-β-Catenin Signaling and Sclerostin on the Phenotypes of Rat Pheochromocytoma PC12 Cells.

Pheochromocytomas and paragangliomas (PPGLs) are classified into 3 major categories with distinct driver genes: pseudohypoxia, kinase signaling, and Wnt-altered subtypes. PPGLs in the Wnt-altered subtype are sporadic and tend to be aggressive with metastasis, where somatic gene fusions affecting mastermind-like 3 (MAML3) and somatic mutations in cold shock domain containing E1 (CSDE1) cause overactivation of Wnt-β-catenin signaling. However, the relation between Wnt-β-catenin signaling and the biological behavior of PPGLs remains unexplored. In rat pheochromocytoma PC12 cells, Wnt3a treatment enhanced cell proliferation and suppressed mRNA expression of tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine biosynthesis, and dopamine secretion. We identified the expression of sclerostin in PC12 cells, which is known as an osteocyte-derived negative regulator for Wnt signaling-driven bone formation. Inhibition of endogenous Wnt pathway by XAV939 or sclerostin resulted in attenuated cell proliferation and increased TH expression. Furthermore, Wnt3a pretreatment suppressed bone morphogenetic protein (BMP)-induced Smad1/5/9 phosphorylation whereas BMPs enhanced sclerostin expression in PC12 cells. In the Wnt-altered subtype, the increased Wnt-β-catenin pathway may contribute the aggressive clinical behavior with reduced catecholamine production. Furthermore, upregulated expression of sclerostin by BMPs may explain the osteolytic metastatic lesions observed in metastatic PPGLs.