Pathway In PC12 Cells Research Articles

Activation of α7 Nicotinic Acetylcholine Receptor Augments Nerve Growth Factor Action on PCtrk Cells

Although cigarette smoking is known to be a critical risk factor for various organ systems and cancers, accumulating evidence indicates that nicotine – a main constituent of cigarette smoking – can exert neuroprotective effects on neuronal cells through nicotinic acetylcholine receptors (nAChRs). However, the precise molecular mechanisms for nicotinic neuroprotective actions remain to be fully elucidated. In this study, we examine the effects of agonists, such as nicotine and PNU282987, on tropomyosin-related kinase (Trk)-dependent neuroprotective pathways in PC12 cells overexpressing a Trk neurotrophin receptor (PCtrk cells). We found that even considerably higher concentrations (mM range for nicotine and µM range for PN282987) of nAChR agonists exert favorable effects, such as the augmentation of nerve growth factor (NGF)-induced Trk neurotrophin receptor autophosphorylation of tyrosine residues and NGF-induced neurite extension. Moreover, nicotine upregulated reactive oxygen species (ROS) levels in the cells. ROS production was completely cancelled by pretreatment with Mito-Tempo, a mitochondria-targeted antioxidant, indicating that the main source of ROS production by nicotine was mitochondria. Furthermore, treatment with nAChR agonists appeared to induce autophagic flux, as evidenced by the upregulation of LC3-II expression in cells. Furthermore, sucrose density ultracentrifugation of nicotine-treated cells clearly disclosed the augmented recruitment of α7nAChR protein into the lipid rafts fraction of the membrane. Intriguingly, a pull-down assay of anti-Trk antibody immunoprecipitates clearly included α7nAChR protein, indicating that Trk and α7nAChR proteins form a complex. These results reveal a new molecular interaction between activated α7nAChR and Trk protein that may serve as a new molecular basis of nicotine-induced neuroprotective action.

Preconditioning of Mesenchymal Stem Cells Enhances the Neuroprotective Effects of Their Conditioned Medium in an Alzheimer's Disease In Vitro Model.

Alzheimer's disease (AD) develops as a result of oxidative damage to neurons and chronic inflammation of microglia. These processes can be influenced by the use of a conditioned medium (CM) derived from mesenchymal stem cells (MSCs). The CM contains a wide range of factors that have neurotrophic, antioxidant, and anti-inflammatory effects. In addition, the therapeutic potential of the CM can be further enhanced by pretreating the MSCs to increase their paracrine activity. The current study aimed to investigate the neuroprotective effects of CM derived from MSCs, which were either activated by a TLR3 ligand or exposed to CoCl2, a hypoxia mimetic (pCM or hCM, respectively), in an in vitro model of AD. We have developed a novel in vitro model of AD that allows us to investigate the neuroprotective and anti-inflammatory effects of MSCs on induced neurodegeneration in the PC12 cell line and the activation of microglia using THP-1 cells. This study demonstrates for the first time that pCM and hCM exhibit more pronounced immunosuppressive effects on proinflammatory M1 macrophages compared to CM derived from untreated MSCs (cCM). This may help prevent the development of neuroinflammation by balancing the M1 and M2 microglial phenotypes via the decreased secretion of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and increased secretion of IL-4, as well as the expression of IL-10 and TGF-β by macrophages. Moreover, a previously unknown increase in the neurotrophic properties of hCM was discovered, which led to an increase in the viability of neuron-like PC12 cells under H2O2-induced oxidative-stress conditions. These results are likely associated with an increase in the production of growth factors, including vascular endothelial growth factor (VEGF). In addition, the neuroprotective effects of CM from preconditioned MSCs are also mediated by the activation of the Nrf2/ARE pathway in PC12 cells. TLR3 activation in MSCs leads to more potent immunosuppressive effects of the CM against pro-inflammatory M1 macrophages, while the use of hCM led to increased neurotrophic effects after H2O2-induced damage to neuronal cells. These results are of interest for the potential treatment of AD with CM from preactivated MSCs.

PLCL/SF/NGF nerve conduit loaded with RGD-TA-PPY hydrogel promotes regeneration of sciatic nerve defects in rats through PI3K/AKT signalling pathways.

Peripheral nerve defect are common clinical problem caused by trauma or other diseases, often leading to the loss of sensory and motor function in patients. Autologous nerve transplantation has been the gold standard for repairing peripheral nerve defects, but its clinical application is limited due to insufficient donor tissue. In recent years, the application of tissue engineering methods to synthesize nerve conduits for treating peripheral nerve defect has become a current research focus. This study introduces a novel approach for treating peripheral nerve defects using a tissue-engineered PLCL/SF/NGF@TA-PPy-RGD conduit. The conduit was fabricated by combining electrospun PLCL/SF with an NGF-loaded conductive TA-PPy-RGD gel. The gel, synthesized from RGD-modified tannic acid (TA) and polypyrrole (PPy), provides growth anchor points for nerve cells. Invitro results showed that this hybrid conduit could enhance PC12 cell proliferation, migration, and reduce apoptosis under oxidative stress. Furthermore, the conduit activated the PI3K/AKT signalling pathway in PC12 cells. In a rat model of sciatic nerve defect, the PLCL/SF/NGF@TA-PPy-RGD conduit significantly improved motor function, gastrocnemius muscle function, and myelin sheath axon thickness, comparable to autologous nerve transplantation. It also promoted angiogenesis around the nerve defect. This study suggests that PLCL/SF/NGF@TA-PPy-RGD conduits provide a conducive environment for nerve regeneration, offering a new strategy for peripheral nerve defect treatment, this study provided theoretical basis and new strategies for the research and treatment of peripheral nerve defect.

Colistin Induces Oxidative Stress and Apoptotic Cell Death through the Activation of the AhR/CYP1A1 Pathway in PC12 Cells.

Colistin is commonly regarded as the "last-resort" antibiotic for combating life-threatening infections caused by multidrug-resistant (MDR) gram-negative bacteria. Neurotoxicity is a potential adverse event associated with colistin application in clinical settings, yet the exact molecular mechanisms remain unclear. This study examined the detrimental impact of colistin exposure on PC12 cells and the associated molecular mechanisms. Colistin treatment at concentrations of 0-400 μM decreased cell viability and induced apoptotic cell death in both time- and concentration-dependent manners. Exposure to colistin triggered the production of reactive oxygen species (ROS) and caused oxidative stress damage in PC12 cells. N-acetylcysteine (NAC) supplementation partially mitigated the cytotoxic and apoptotic outcomes of colistin. Evidence of mitochondrial dysfunction was observed through the dissipation of membrane potential. Additionally, colistin treatment upregulated the expression of AhR and CYP1A1 mRNAs in PC12 cells. Pharmacological inhibition of AhR (e.g., using α-naphthoflavone) or intervention with the CYP1A1 gene significantly decreased the production of ROS induced by colistin, subsequently lowering caspase activation and cell apoptosis. In conclusion, our findings demonstrate, for the first time, that the activation of the AhR/CYP1A1 pathway contributes partially to colistin-induced oxidative stress and apoptosis, offering insights into the cytotoxic effects of colistin.

Evidence of Urtica dioica Agglutinin's Antiproliferative and Anti-migratory Potentials on the Hyaluronic Acid-Overexpressing Prostate Cancer Cells.

Hyaluronic acid is composed of repeating sugar units, glucuronic acid and N-acetylglucosamine, which are often associated with increased tumor progression. Urtica dioica agglutinin is a potential component that exhibits a high affinity for binding to N-acetylglucosamine. This study aimed to investigate U. dioica Agglutinin's potential to inhibit the proliferation and migration of prostate cancer cells with high expression of hyaluronic acid through molecular docking and in vitro studies. The expression of hyaluronan synthase genes in prostate tissue and cell lines was checked by an in silico study, and the interaction between hyaluronic acid with both CD44 transmembrane glycoprotein and U. dioica agglutinin was analyzed through molecular docking. U. dioica Agglutinin's effect on cell viability (neutral red uptake assay), migration (scratch wound healing assays), and both CD44 and Nanog expression (quantitative real-time polymerase chain reaction) were assessed in vitro. The results showed that in prostate cancer cell lines, the PC3 cell line has the highest expression of hyaluronan synthase genes. U. dioica agglutinin exhibits an interaction of six specific residues on CD44 compared to hyaluronic acid's singular residue. While U. dioica agglutinin alone effectively reduced cell viability and wound closer (≥ 150 µg/mL), combining it with hyaluronic acid significantly shifted the effective concentration to a higher dose (≥ 350 µg/mL). These results, together with low Nanog and high CD44 gene expression, suggest that U. dioica agglutinin may impair the CD44-HA pathway in PC3 cells. This possibility is supported by U. dioica Agglutinin's ability to compete with hyaluronic acid for binding to CD44. Based on this, U. dioica agglutinin as a plant lectin shows promise in inhibiting cancer proliferation and migration by targeting its dependence on hyaluronic acid.

Combination of navitoclax (Bcl-2 and Bcl-xL inhibitor) and Debio-0932 (Hsp90 inhibitor) suppresses the viability of prostate cancer cells via induction of apoptotic signaling pathway.

Prostate cancer is one of the most common cancers in men. Given the diverse nature of prostate cancer and its tendency to respond differently to various treatments, combination therapies are often employed to enhance outcomes. In this study, the synergetic efficiency of chemotherapeutic drug Navitoclax and heat shock protein 90 (Hsp90) inhibitor Debio-0932 was evaluated in human prostate cancer cell line (PC3). Our results indicated that Navitoclax-Debio-0932 combination exhibited synergistic activity in PC3 cells at concentrations lower than IC50 values. The combination of Navitoclax and Debio-0932 decreased PC3 cell viability in a dose dependent manner at 48h. To investigate the apoptotic potential of the Navitoclax-Debio-0932 combination against prostate cancer cells, the mRNA and protein expression levels of apoptotic and antiapoptotic markers (Bax, Bcl-2, Bcl-xL, Cyt-c, Apaf-1, Casp-3, Casp-7, and Casp-9) were measured using RT-PCR and ELISA assay. Furthermore, the cleavage activity of Casp-3 was determined by colorimetric assay. The results revealed that Navitoclax-Debio-0932 combination potently induced intrinsic apoptotic pathway in PC3 cells rather than using drugs alone. The combined treatment of Navitoclax and Debio-0932 displayed synergistic cytotoxic and apoptotic effects on prostate cancer cells, presenting a promising approach for combination therapy in prostate cancer.

Exploring the mechanism of action of Sparganii Rhizoma-Curcumae Rhizoma for in treating castration-resistant prostate cancer: a network-based pharmacology and experimental validation study

Sparganii Rhizoma-Curcumae Rhizoma (SR-CR) is a classic drug pair for the treatment of castration-resistant prostate cancer (CRPC), but its mechanism has not been clarified. The study aims to elucidate the potential mechanism of SR-CR in the management of CRPC. The present study employed the TCMSP as well as the SwissTargetPrediction platform to retrieve the chemical composition and targets of SR-CR. The therapeutic targets of CRPC were identified through screening the GeneCards, Disgenet, and OMIM databases. Subsequently, the Venny online platform was utilized to identify the shared targets between the SR-CR and CRPC. The shared targets were enrichment analysis using the Bioconductor and Kyoto encyclopedia of genes and genomes (KEGG) databases. The active ingredients and core targets were verified through molecular docking and were validated using PC3 cells in the experimental validation phase. A total of 7 active ingredients and 1126 disease targets were screened from SR-CR, leading to a total of 59 shared targets. Gene Ontology (GO) analysis resulted in 1309 GO entries. KEGG pathways analysis yielded 121 pathways, primarily involving cancer-related signaling pathways. The results from molecular docking revealed stable binding interactions between the core ingredients and the core targets. In vitro cellular assays further demonstrated that SR-CR effectively suppressed the activation of the Prostate cancer signaling pathway in PC3 cells, leading to the inhibition of cell proliferation and promotion of apoptosis. The SR-CR exert therapeutic effects on CRPC by inhibiting cell proliferation and promoting apoptosis through the Prostate cancer signaling pathway.

Dieckol prevents prostate cancer cell proliferation by transcriptionally attenuating JAK/STAT3 signaling pathway.

This study delved at how the natural substance dieckol (DCL) prevents prostate cancerous cells from proliferating and migrating by blocking the JAK/STAT3 signaling pathway in PC-3 cells. For numerous tests, the cells were treated to DCL at a range of concentrations (0-20 μM) for 24 h. DCL mediated cytotoxicity was analyzed by MTT assay. To evaluate ROS, DCFH-DA staining was employed. Dual (AO/EtBr) staining was utilized to examine apoptotic changes, and MMP levels in PC-3 cells were examined using the appropriate fluorescent staining assays. By using flow cytometry and western blotting, the protein expressions of cell survival, cell cycle, proliferation, and apoptosis were assessed. The results showed that DCL significantly cytotoxically affects PC-3, and the IC50 was discovered to be 12 μM for 24 h exposure. Furthermore, after DCL treatment in PC-3, considerable ROS generation and increased apoptotic signals were detected. STAT3, JAK1, PCNA, and cyclins D1 and E1 are all suppressed by DCL in PC-3. In addition, DCL therapy in PC-3 dramatically increased pro-apoptotic proteins such Bax, caspase-3, and cytochrome C. Therefore, DCL has been regarded as a chemotherapeutic agent because to its ability to decrease the expression of proteins that control cell proliferation, including STAT3, JAK1, PCNA, and cyclins D1 and E1.

Lactic Acid Bacteria-Derived Exopolysaccharides Mitigate the Oxidative Response via the NRF2-KEAP1 Pathway in PC12 Cells.

Parabiotics, including L-EPSs, have been administered to patients with neurodegenerative disorders. However, the antioxidant properties of L-EPSs against H2O2-induced oxidative stress in PC12 cells have not been studied. Herein, we aimed to investigate the antioxidant properties of the L-EPSs, their plausible targets, and their mechanism of action. We first determined the amount of L-EPSs in Lactobacillus delbrueckii ssp. bulgaricus B3 and Lactiplantibacillus plantarum GD2 using spectrophotometry. Afterwards, we studied their effects on TDH, TOS/TAS, antioxidant enzyme activities, and intracellular ROS level. Finally, we used qRT-PCR and ELISA to determine the effects of L-EPSs on the NRF2-KEAP1 pathway. According to our results, the L-EPS groups exhibited significantly higher total thiol activity, native thiol activity, disulfide activity, TAS levels, antioxidant enzyme levels, and gene expression levels (GCLC, HO-1, NRF2, and NQO1) than did the H2O2 group. Additionally, the L-EPS groups caused significant reductions in TOS levels and KEAP1 gene expression levels compared with those in the H2O2 group. Our results indicate that H2O2-induced oxidative stress was modified by L-EPSs. Thus, we revealed that L-EPSs, which regulate H2O2-induced oxidative stress, could have applications in the field of neurochemistry.

The Neuroprotective Effects of Flavonoid Fisetin against Corticosterone-Induced Cell Death through Modulation of ERK, p38, and PI3K/Akt/FOXO3a-Dependent Pathways in PC12 Cells.

The overactive hypothalamic-pituitary-adrenal (HPA) axis is believed to trigger the overproduction of corticosterone, leading to neurotoxicity in the brain. Fisetin is a flavonoid commonly found in fruits and vegetables. It has been suggested to possess various biological activities, including antioxidant, anti-inflammatory, and neuroprotective effects. This study aims to explore the potential neuroprotective properties of fisetin against corticosterone-induced cell death and its underlying molecular mechanism in PC12 cells. Our results indicate that fisetin, at concentrations ranging from 5 to 40 μM, significantly protected PC12 cells against corticosterone-induced cell death. Fisetin effectively reduced the corticosterone-mediated generation of reactive oxygen species (ROS) in PC12 cells. Fisetin treatments also showed potential in inhibiting the corticosterone-induced apoptosis of PC12 cells. Moreover, inhibitors targeting MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK, and phosphatidylinositol 3-kinase (PI3K) were found to significantly block the increase in cell viability induced by fisetin in corticosterone-treated cells. Consistently, fisetin enhanced the phosphorylation levels of ERK, p38, Akt, and c-AMP response element-binding protein (CREB) in PC12 cells. Additionally, it was found that the diminished levels of p-CREB and p-ERK by corticosterone can be restored by fisetin treatment. Furthermore, the investigation of crosstalk between ERK and CREB revealed that p-CREB activation by fisetin occurred through the ERK-independent pathway. Moreover, we demonstrated that fisetin effectively counteracted the corticosterone-induced nuclear accumulation of FOXO3a, an apoptosis-triggering transcription factor, and concurrently promoted FOXO3a phosphorylation and its subsequent cytoplasmic localization through the PI3K/Akt pathway. In conclusion, our findings indicate that fisetin exerts its neuroprotective effect against corticosterone-induced cell death by modulating ERK, p38, and the PI3K/Akt/FOXO3a-dependent pathways in PC12 cells. Fisetin emerges as a promising phytochemical for neuroprotection.

Overexpression of FSP1 Ameliorates ferroptosis via PI3K/ AKT /GSK3β pathway in PC12 cells with Oxygen-Glucose Deprivation/Reoxygenation

After ischemia and reperfusion (I/R), nerve cell damage is a pathogenic process that involves numerous molecular processes. In the last ten years, one new classification of programmed cell death is ferroptosis. More recent research has demonstrated that ferroptosis has a role in a variety of neurological disorders, including stroke, cancer, and neurodegenerative illnesses. Ferroptosis suppressor protein 1 (FSP1) plays a significant role in inhibiting ferroptosis. The purpose of this work is to determine how overexpression of FSP1 affects the ferroptosis of PC12 cells under the condition of oxygen-glucose deprivation/reoxygenation (OGD/R). The expression of FSP1 was regulated by lentivirus transfection technology. Western blot and immunofluorescence were used to measure protein levels related to ferroptosis and the PI3K/AKT/GSK3β signal pathway. Determine cell viability using the appropriate kit. Mitochondrial structural morphology was checked by transmission electron microscopy in PC12 cells. Reactive oxygen species (ROS) and Malondialdehyde (MDA) were quantified using the relevant kits. OGD/R induced ferroptosis in PC12 cells, however, FSP1 overexpression reverses ferroptosis and promotes cell viability, lowering ROS and MDA content. The expression of FSP1 decreased in OGD/R0h and OGD/R6h and rebounded in OGD/R24h and OGD/R48h. During the processes of OGD/R-induced ferroptosis, FSP1 overexpression significantly stimulated PI3K/AKT/GSK3β pathway, but LY294002 weakens the protective effect of FSP1 overexpression. Our outcomes demonstrate that overexpression of FSP1 markedly enhances the ability to resist ferroptosis via the PI3K/AKT/GSK3β pathway. The above results may provide a new preliminary lead for the treatment of the cerebral ischemia-reperfusion injury.

HSF1 Alleviates Brain Injury by Inhibiting NLRP3-Induced Pyroptosis in a Sepsis Model.

Sepsis, which could cause a systemic inflammatory response, is a life-threatening disease with a high morbidity and mortality rate. There is evidence that brain injury may be related to severe systemic infection induced by sepsis. The brain injury caused by sepsis could increase the risk of mortality in septic patients, which seriously affects the septic patient's prognosis of survival. Although there remains a focus on sepsis research, clinical measures to prevent and treat brain injury in sepsis are not yet available, and the high mortality rate is still a big health burden. Therefore, it is necessary to investigate the new molecules or regulated pathways that can effectively inhibit the progress of sepsis. NLR family pyrin domain-containing 3 (NLRP3) increased in the procession of sepsis and functioned as the key regulator of pyroptosis. Heat shock factor 1 (HSF1) can protect organs from multiorgan dysfunction syndrome induced by lipopolysaccharides in mice, and NLRP3 could be inhibited by HSF1 in many organs. However, whether HSF1 regulated NLRP3 in sepsis-induced brain injury, as well as the detailed mechanism of HSF1 in brain injury, remains unknown in the sepsis model. In this research, we try to explore the relationship between HSF1 and NLRP3 in a sepsis model and try to reveal the mechanism of HSF1 inhibiting the process of brain injury. In this study, we used wild-type mice and hsf1 -/- mice for in vivo research and PC12 cells for in vitro research. Real-time PCR and Western blot were used to analyze the expression of HSF1, NLRP3, cytokines, and pyrolytic proteins. EthD-III staining was chosen to detect the pyroptosis of the hippocampus and PC12 cells. The results showed that HSF1 is negatively related to pyroptosis. The pyroptosis in cells of brain tissue was significantly increased in the hsf1 -/- mouse model compared to hsf1 +/+ mice. In PC12 cells, hsf1 siRNA can upregulate pyroptosis while HSF1-transfected plasmid could inhibit the pyroptosis. HSF1 could negatively regulate the NLRP3 pathway in PC12 cells, while hsf1 siRNA enhanced the pyroptosis in PC12 cells, which could be reversed by nlrp3 siRNA. These results imply that HSF1 could alleviate sepsis-induced brain injury by inhibiting pyroptosis through the NLRP3-dependent pathway in brain tissue and PC12 cells, suggesting HSF1 as a potential molecular target for treating brain injury in sepsis clinical studies.

Mild hypothermia alleviates oxygen-glucose deprivation/reperfusion-induced apoptosis by inhibiting ROS generation, improving mitochondrial dysfunction and regulating DNA damage repair pathway in PC12 cells.

The brain ischemia/reperfusion (I/R) injury has a great impact on human life and property safety. As far as we know, mild hypothermia (MH) is an effective measure to reduce neuronal injury after I/R. However, the precise mechanism is not extremely clear. The purpose of this study was to investigate whether mild therapeutic hypothermia can play a protective role in nerve cells dealing with brain I/R injury and explore its specific mechanism in vitro. A flow cytometer, cell counting kit-8 (CCK-8) assay and lactate dehydrogenase (LDH) release assay were performed to detect apoptotic rate of cells, cell viability and cytotoxicity, respectively, reactive oxygen species (ROS) assay kit, JC-1 fluorescent methods, immunofluorescence and western blot were used to explore ROS, mitochondrial transmembrane potential (Δψm), mitochondrial permeability transition pore (MPTP) and protein expression, respectively. The result indicated that the cell activity was decreased, while the cytotoxicity and apoptosis rate were increased after treating with oxygen-glucose deprivation/reperfusion (OGD/R) in PC12 cells. However, MH could antagonize this phenomenon. Interestingly, treating with OGD/R increased the release of ROS and the transfer of Cytochrome C (Cyt-C) from mitochondria to cytoplasm. In addition, it up-regulated the expression of γH2AX, Bax and Clv-caspase3, down-regulated the expression of PCNA, Rad51 and Bcl-2, and inhibited the function of mitochondria in PC12 cells. Excitingly, the opposite trend was observed after MH treatment. Therefore, our results suggest that MH protects PC12 cells against OGD/R-induced injury with the mechanism of inhibiting cell apoptosis by reducing ROS production, improving mitochondrial function, reducing DNA damage, and enhancing DNA repair.

Ulinastatin ameliorates the malignant progression of prostate cancer cells by blocking the RhoA/ROCK/NLRP3 pathway.

Prostate cancer is a male malignant tumor disease with high incidence and mortality. This study was designed to explore the effects of ulinastatin (UTI) on the malignant progression of prostate cancer and its relevant mechanism of action.Human prostate cancer cell line PC-3 was applied to investigate the anticancer activity of UTI. PC-3 cells were treated with increasing concentrations (400, 800, and 1600 U/ml) of UTI. Cell proliferation, migration, invasion, and apoptosis were determined by cell counting kit-8 (CCK-8), colony formation, wound-healing, Transwell assay, and flow cytometry analysis, respectively. The expression level of corresponding proteins was detected by western blot. In addition, PC-3 cells were pretreated with RhoA agonist CN03 (1 μg/ml) or NLRP3 agonist nigericin (10 μM)before UTI treatment, and the cellular behaviors above were detected again.It was demonstrated that UTI significantly suppressed cell proliferation, migration, and invasion but promoted apoptosis in PC-3 cells in a concentration-dependent manner. Meanwhile, UTI could block RhoA/ROCK/NLRP3 inflammasome pathway in PC-3 cells, and the activation of RhoA or NLRP3 inflammasome partly weakened the impacts of UTI on cell proliferation, migration, and apoptosis in PC-3 cells, respectively.In summary, our study demonstrated the antitumor activity of UTI against prostate cancer by regulating RhoA/NLRP3 inflammasome pathway, providing a promising candidate drug for the therapeutic treatment of prostate cancer.

Acetylation Enhances the Anticancer Activity and Oral Bioavailability of 5-Demethyltangeretin.

A kind of hydroxylated polymethoxyflavone (PMFs) existing in the citrus genus, 5-Demethyltangeretin (5-DTAN), has been reported to possess several bioactivities in vitro and in vivo. The aim of this study was to investigate whether acetylation could enhance the anticancer activity and oral bioavailability of 5-DTAN. PC-3 human prostate cancer cells were treated with tangeretin (TAN), 5-DTAN, and 5-acetylated TAN (5-ATAN), and the results showed that the cytotoxic effect 5-ATAN (IC50 value of 5.1 µM) on the cell viability of PC-3 cells was stronger than that of TAN (IC50 value of 17.2 µM) and 5-DTAN (IC50 value of 11.8 µM). Compared to 5-DTAN, 5-ATAN treatment caused a more pronounced DNA ladder, increased the sub-G1 phase population, and induced G2/M phase arrest in the cell cycle of PC-3 cells. We also found that 5-ATAN triggered the activation of caspase-3 and the progression of the intrinsic mitochondrial pathway in PC-3 cells, suggesting the induction of apoptosis. In a cell wound healing test, 5-ATAN dose-dependently reduced the cell migration, and the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) was decreased after 48 h of 5-ATAN treatment. Moreover, oral administration of 5-ATAN showed a significantly stronger inhibitory effect on tumor size and tumor weight in tumor-bearing nude mice than those of vehicle or the 5-DTAN group (p < 0.05). Furthermore, pharmacokinetic results showed that single-dose oral administration of 5-ATAN exhibited a higher maximum concentration (Cmax) and area under the curve (AUC) of 5-DTAN in plasma than that of 5-DTAN. More extensive distribution of 5-DTAN to most tissues of mice was also observed in mice treated with 5-ATAN for 7 days. In conclusion, acetylation strongly enhances the anticancer activity and oral bioavailability of 5-DTAN and could be a promising strategy to promote the potential bioactivities of natural products.

Neuroprotection of resveratrol against cadmium-poisoning acts through dual inhibition of mTORC1/2 signaling

Resveratrol is a natural polyphenol with neuroprotective function. The underlying mechanism is not well understood. Our previous studies have identified that resveratrol antagonizes cadmium (Cd) neurotoxicity via targeting PP2A/PP5-mediated Erk1/2 and JNK pathways. Here we show that resveratrol protected against Cd-poisoning also by blocking Cd-induced activation of mTORC1 and mTORC2 pathways in PC12 cells and murine primary neurons. Co-treatment with inhibitors of mTORC1 (rapamycin), mTORC1/2 (PP242), Erk1/2 (U0126) and/or JNK (SP600125), knockdown of mTOR, or disruption of mTORC1 and/or mTORC2 by silencing raptor, rictor or raptor/rictor, respectively, markedly potentiated the inhibitory effects of resveratrol on Cd-induced phosphorylation of S6K1/4E-BP1 (mTORC1 substrates), Akt (mTORC2 substrate), Erk1/2 and/or JNK/c-Jun, cleavage of caspase-3 and cell death in PC12 cells and/or primary neurons. Knockdown of S6K1 or 4E-BP1, or ectopic expression of constitutively hypophosphorylated 4E-BP1 (4E-BP1-5A) reinforced the resveratrol's inhibition on Cd-evoked cell death, whereas ectopic expression of constitutively active S6K1 or knockdown of 4E-BP1 attenuated the resveratrol's inhibition on Cd-induced cell death. Co-treatment with Akt inhibitor or overexpression of dominant negative Akt (dn-Akt) strengthened the resveratrol's suppression on Cd-induced ROS, Erk1/2 activation and apoptosis, whereas overexpression of constitutively active Akt (myr-Akt) conferred high resistance to the resveratrol's inhibitory effects in the neuronal cells. Taken together, the results indicate that resveratrol attenuates Cd-induced neuronal apoptosis partly through inhibition of mTORC1/2 pathways. Our studies highlight that resveratrol can be exploited for the prevention of Cd toxicity related to neurodegenerative diseases.

Tigliane and rhamnofolane glycosides from Euphorbia wallichii prevent oxidative stress-induced neuronal death in PC-12 cells

Tigliane and rhamnofolane diterpenoids bearing glycosyl substituents are rarely found in nature. In the current study, seven new tigliane glycosides, euphorwallsides A − G (1–7), and five new rhamnofolane glycosides, euphorwallsides H − L (8–12), were isolated from the whole plants of Euphorbia wallichii. Their structures were elucidated by a combination of spectroscopic, computational, and chemical means. The aglycones of 1–5 represent a rare class of 13-deoxygenated tiglianes, while those of 8–12 represent the first examples of 4-deoxygenated rhamnofolanes. 2, 3, 5, 7, 8, and 12 showed significant neuroprotective effects on sodium nitroprusside (SNP)-induced neuronal death in pheochromocytoma cell line PC-12 at 10 μM, being more active than the clinical drug, edaravone. Mechanistic study revealed that the most active compound, 3, could inhibit reactive oxygen species (ROS) accumulation and restore the mitochondrial membrane potential via modulating the Nrf2 signaling pathway in PC-12 cells.

Microwave assisted extraction, characterization of a polysaccharide from Salvia miltiorrhiza Bunge and its antioxidant effects via ferroptosis-mediated activation of the Nrf2/HO-1 pathway

A microwave-assisted extraction procedure for the crude Salvia miltiorrhiza polysaccharides (SMPs) obtained from Salvia miltiorrhiza Bunge was optimized. Four independent variables were studied: microwave power, extraction time, solvent-to-solid ratio, and concentration of ethanol, with optimal settings of 1200 W, 12 min, 38, and 86 %, respectively. The SMPs were successively purified by DEAE Sepharose Fast Flow and Sephadex G-100 chromatography to produce a novel polysaccharide termed SMP1. The SMP1 was composed of glucose, galactose, and fructose in a molar ratio of 1:1.67:1.12 with an average molecular weight of 6087 Da. Pharmacological studies showed that SMP1 protected from OGD/R-induced ferroptosis and lipid peroxidation by activating Nrf2/HO-1 pathway in PC12 cells. Our research systematically indicated that polysaccharide could inhibit ferroptosis to alleviate oxidative stress injury, which laid the foundation for the future clinical application of Salvia miltiorrhiza polysaccharide.

Hispolon alleviates oxidative damage by stimulating the Nrf2 signaling pathway in PC12 cells

Natural products derived from the daily diet are garnering increasing attention for neurodegenerative disease (ND) treatment. Hispolon (His), a small molecule from Phellinus linteus, has been reported to have various pharmacological activities. Here, we evaluated its protective effect on a neuron-like rat pheochromocytoma cell line (PC12). Results showed that His could restore cell death induced by oxidative damage. Nuclear factor-erythroid 2 (NF-E2)-related factor 2 (Nrf2) plays a significant role in maintaining cellular redox homeostasis. After treatment with His, some Nrf2-governed antioxidant genes were upregulated in a dose-dependent manner. However, the protective effect of His on PC12 cells was easily terminated by Nrf2 knockdown, demonstrating that Nrf2 is a critical component in this cytoprotective process. Taken together, our study showed that His was not only an effective activator of Nrf2 but also a promising candidate for ND treatment.

Learning and memory impairment induced by 1,4-butanediol is regulated by ERK1/2-CREB-BDNF signaling pathways in PC12 cells.

1,4-butanediol (1,4-BD) is a known γ-hydroxybutyric acid (GHB) precursor which affects the nervous system after ingestion, leading to uncontrolled behavioral consequences. In the present study, we investigated whether 1,4-BD induces oxidative stress and inflammation in PC12 cells and evaluated the toxic effects of 1,4-BD associates with learning and memory. CCK-8 results revealed a dose-effect relationship between the cell viability of PC12 cells and 1,4-BD when the duration of action was 2h or 4h. Assay kits results showed that 1,4-BD decreased the levels of Glutathione (GSH), Glutathione peroxidase (GSH-px), Superoxide dismutase (SOD), Acetylcholine (Ach) and increased the levels of Malondialdehyde (MDA), Nitric oxide (NO) and Acetylcholinesterase (AchE). Elisa kits results indicated that 1,4-BD decreased the levels of synaptophysin I (SYN-1), Postsynaptic density protein-95 (PSD-95), Growth associated protein-43 (GAP-43) and increased the levels of Tumor necrosis factor alpha (TNF-α) and Interleukin- 6 (IL-6). RT-PCR results showed that the mRNA levels of PSD-95, SYN-1 and GAP-43 were significantly decreased. The expression of phosphorylation extracellular signal-regulated protein kinase 1/2 (p-ERK1/2), phosphorylation cAMP response element binding protein (p-CREB) and brain-derived neurotrophic factor (BDNF) proteins were significantly decreased in PC12 cells by protein blotting. Overall, these results suggest that 1,4-BD may affect synaptic plasticity via the ERK1/2-CREB-BDNF pathway, leading to Ach release reduction and ultimately to learning and memory impairment. Furthermore, oxidative stress and inflammation induced by 1,4-BD may also result in learning and memory deficits. These findings will enrich the toxicity data of 1.4-BD associated with learning and memory impairment.