Induced Injury In PC12 Cells Research Articles

RNA-seq revealed the protective effect of Huangqi Guizhi Wuwu Decoction against cisplatin induced PC12 cell injury

Background Chemotherapy-induced peripheral neuropathy (CIPN) not only affects the tolerability of chemotherapy, but also causes intolerable and prolonged neuropathic pain in cancer patients. Currently, duloxetine is the only drug used to treat CIPN. However, the clinical use of this drug still faces several challenges. Therefore, we focused on traditional Chinese medicine (TCM) to find an effective and safe alternative medicine. Huangqi Guizhi Wuwu Decoction (HGWD) is a traditional Chinese medicine that has been clinically used for treating nerve pain for thousands of years. This study aimed to investigate the neuroprotective effect of HGWD on cisplatin-induced nerve injury in PC12 cells and to elucidate its potential mechanism of action. Methods HGWD-containing serum and blank serum were prepared from a rat model. The protective effects of HGWD on cisplatin (10 µmol/L)-induced PC12 cell injury were assessed by a Cell Counting Kit-8 (CCK-8) assay. RNA expression in HGWD-protected PC12 cells was analyzed using RNA-seq, and subsequently, differentially expressed genes (DEGs) were further analyzed using Gene Ontology (GO) and Gene Set Enrichment Analysis (GSEA). Results The CCK-8 results showed that pretreatment of PC12 cells with HGWD-containing serum (5%, 10%, 15%) significantly increased cells' viability to 10 µmol/L cisplatin-induced cell death. RNA-seq analysis revealed 843 DEGs in the CIPN group and 249 in the HGWD group. The GSEA results in this study suggest that HGWD may treat CIPN by enhancing axon guidance. Conclusions This study provides valuable evidence for using HGWD in treating CIPN, partially achieved by improving axon guidance pathways.

Investigation into Antioxidant Mechanism of Lycium barbarum Extract in Carbendazim-Induced PC12 Cell Injury Model through Transcriptomics and Metabolomics Analyses.

Lycium barbarum L., an important functional food in China, has antioxidant and antiaging activity. However, the exact antioxidant activity mechanism of Lycium barbarum extracts (LBE) is not well understood. Therefore, a carbendazim (CBZ)-induced PC12 cell injury model was constructed and vitrificated to study the antioxidant activity of fresh LBE on the basis of extraction parameter optimization via the full factorial design of experiments (DOE) method. The results showed that the pretreatment of PC12 cells with LBE could reduce the reactive oxygen species (ROS) level by 14.6% and inhibited the mitochondrial membrane potential (MMP) decline by 12.0%. Furthermore, the integrated analysis revealed that LBE played an antioxidant role by activating oxidative phosphorylation (OXPHOS) and restoring MMP, maintaining the tricarboxylic acid (TCA) cycle stability, and regulating the GSH metabolic pathway. The results of the present study provide new ideas for the understanding of the antioxidant function of LBE from a global perspective.

Identification of antidepressant constituents from Xiangfu-chuanxiong herbal medicine pair via spectrum-effect relationship analyses, molecular docking and corticosterone-induced PC12 cells

Herbal medicine pair, composed of two single herbs, is a relatively fixed minimum prescription unit in the traditional Chinese medicine's formula and has special significance in clinic. The combination of Xiangfu (the rhizoma of Cyperus rotundus L, XF) and Chuanxiong (the rhizoma of Ligusticum chuanxiong Hort, CX) has been recoded as an herbal medicine pair XF-CX in the Yuan Dynasty (1347 CE) of China and widely used in traditional Chinese medicine formula, including Chaihu Shugan San, which has been clinically used for treatment of depression. However, the optimal ratio of the XF-CX herbal medicine pair and its antidepressant constituents are still unclear. Herein, the antidepressive-like effects of XF-CX herbal medicine pairs with different ratios of XF and CX (2:1, 1:1, 1:2) were evaluated using behavioral despair animal models in mice, and then its potential antidepressant constituents were recognized by spectrum-effect relationship analyses. Finally, the potential antidepressant constituents of the XF-CX herbal medicine pair were validated by molecular docking with glucocorticoid receptor and corticosterone (CORT)-induced PC12 cell injury model. The results indicated that different ratios of XF-CX pairs had antidepressive-like effects, and the XF-CX (2:1) exhibited a more significant effect. Thirty-two potential antidepressant constituents in the XF-CX herbal medicine pair were screened out from the spectrum-effect relationship combined molecular docking analyses. Among them, senkyunolide A, cyperotundone, Z-ligustilide, and levistilide A were validated to have protective effects against CORT-induced injury in PC12 cells. Our findings not only obtained the optimal ratio of XF-CX in the herbal medicine pair for the treatment of depression but also its potential antidepressant constituents, which will benefit in elucidating the mechanism of action and promoting the application of the herbal medicine pair in the clinic.

The Activation of GABAAR Alleviated Cerebral Ischemic Injury via the Suppression of Oxidative Stress, Autophagy, and Apoptosis Pathways.

Ischemic stroke is a devastating disease leading to neurologic impairment. Compounding the issue is the very limited array of available interventions. The activation of a γ-aminobutyric acid (GABA) type A receptor (GABAAR) has been reported to produce neuroprotective properties during cerebral ischemia, but its mechanism of action is not yet fully understood. Here, in a rat model of photochemically induced cerebral ischemia, we found that muscimol, a GABAAR agonist, modulated GABAergic signaling, ameliorated anxiety-like behaviors, and attenuated neuronal damage in rats suffering cerebral ischemia. Moreover, GABAAR activation improved brain antioxidant levels, reducing the accumulation of oxidative products, which was closely associated with the NO/NOS pathway. Notably, the inhibition of autophagy markedly relieved the neuronal insult caused by cerebral ischemia. We further established an oxygen-glucose deprivation (OGD)-induced PC12 cell injury model. Both in vivo and in vitro experiments demonstrated that GABAAR activation obviously suppressed autophagy by regulating the AMPK-mTOR pathway. Additionally, GABAAR activation inhibited apoptosis through inhibiting the Bax/Bcl-2 pathway. These data suggest that GABAAR activation exerts neuroprotective effects during cerebral ischemia through improving oxidative stress and inhibiting autophagy and apoptosis. Our findings indicate that GABAAR serves as a target for treating cerebral ischemia and highlight the GABAAR-mediated autophagy signaling pathway.

Kaempferol-3-O-sophoroside (PCS-1) contributes to modulation of depressive-like behaviour in C57BL/6J mice by activating AMPK.

Kaempferol-3-O-sophoroside (PCS-1) is the main component in Crocus sativus (Saffron), a herb with mood-enhancing properties. AMP-activated protein kinase (AMPK) is a potential therapeutic target for depression. This study explores the antidepressive-like properties of PCS-1 and its AMPK activation to confirm AMPK as a target for antidepression. Corticosterone (CORT)-induced PC12 cell injury served as an in vitro model to evaluate the neuroprotective effect of PCS-1. Neuro-2a cells and primary neurons were utilized to evaluate the synaptogenesis role of PCS-1. CORT-induced mouse depression model and chronic unpredictable mild stress (CUMS) model were used to assess the antidepressive-like properties of PCS-1 through behavioural tests, magnetic resonance imaging, and biochemical index measurements. Western blot and immunofluorescence assays were used to study the mechanisms of PCS-1. Cellular thermal shift assay was used to confirm the binding target. PCS-1 (12.5-50 μM) ameliorated CORT-induced PC12 cell damage, oxidative stress and inflammation. PCS-1 alone promoted an increase in synapses in Neuro-2a cells and primary neurons. Oral administration of PCS-1 (10 and 20 mg·kg-1 ) ameliorated weight loss, dyskinesia, and hippocampal volume reduction induced by CORT and CUMS. PCS-1 bound to AMPK to improve the expression of brain-derived neurotrophic factor (BDNF) and induce autophagy. PCS-1 binds to AMPK to promote BDNF production and autophagy enhancement, ultimately achieving antidepressant effects. This study provides support for the clinical application of saffron petals and provides further evidence for AMPK as a potential target for antidepression.

Highly oxidized and rearranged schinortriterpenoids with neuroprotective activity from the stems and leaves of Schisandra chinensis

The stems and leaves (SCSL) are the main byproducts of the Schisandra chinensis cultivation process, and they are worthy of research and utilization from the perspective of medicine and economic benefits. Eighteen (1–18) undescribed highly oxygenated and rearranged schinortriterpenoids (SNTs) and six analogues (19–24), including 14(13 → 12):16(17 → 13)-diabeoschiartane (1–7), 18(13 → 14)-abeoschiartanes (8–12, 19), 18-norschiartane (13), schiartanes (14, 20), 16,17-secopreschisanartanes (15–17, 21–24), lancifoartane (18) skeletons, were isolated from the SCSL. Compounds 1–7 feature a rare 7/5/5-fused carbocyclic core. The structures were established by MS, NMR, single-crystal X-ray diffraction, ECD, and biogenetic considerations. In addition, neuroprotective assays were performed to gain a preliminary understanding of their biological activity. Compounds 2, 6, and 7 showed strong neurite outgrowth-promoting activity with 13.1 %, 12.0 %, and 12.2 % cell differentiation rate (positive group: 15.4 %), respectively. Compounds 1, 2, and 11 at a concentration of 25 μM also had neuroprotective effect on corticosterone (CORT)-induced PC12 cell injury, the cell viability was increased by 21.1 %, 19.5 %, and 24.4 % (positive group: 30.6 %), respectively. Molecular docking analysis and protein–protein interaction (PPI) network revealed that compounds 2, 6, and 7 can exert neuroprotective effects through the regulation of growth factor receptor-bound protein 2 (GRB2). The discovery was beneficial to the high-value utilization of SCSL and the development of natural neuroprotective drugs.

Tetramethylpyrazine Attenuates Oxygen-glucose Deprivation-induced Neuronal Damage through Inhibition of the HIF-1α/BNIP3 Pathway: from Network Pharmacological Finding to Experimental Validation

A network pharmacological analysis combined with experimental validation was used to investigate the neuroprotective mechanism of the natural product Tetramethylpyrazine(TMP). Protecting neurons is critical for acute ischemic stroke treatment. Tetramethylpyrazine is a bioactive component extracted from Chuanxiong. The neuroprotective potential of TMP has been reported, but a systematic analysis of its mechanism has not been performed. Based on the hints of network pharmacology and bioinformatics analysis, the mechanism by which TMP alleviates oxygen-glucose deprivation-induced neuronal damage through inhibition of the HIF-1α/BNIP3 pathway was verified. In this study, we initially used network pharmacology and bioinformatics analyses to elucidate the mechanisms involved in TMP's predictive targets on a system level. The HIF-1α/BNIP3 pathway mediating the cellular response to hypoxia and apoptosis was considered worthy of focus in the bioinformatic analysis. An oxygen-glucose deprivation (OGD)-induced PC12 cell injury model was established for functional and mechanical validation. Cell viability, lactate dehydrogenase leakage, intracellular reactive oxygen species, percentage of apoptotic cells, and Caspase-3 activity were determined to assess the TMP's protective effects. Transfection with siRNA/HIF-1α or pcDNA/HIF-1α plasmids to silence or overexpress hypoxia-inducible factor 1α(HIF-1α). The role of HIF-1α in OGD-injured cells was observed first. After that, TMP's regulation of the HIF-1α/BNIP3 pathway was investigated. The pcDNA3.1/HIF-1α-positive plasmids were applied in rescue experiments. The results showed that TMP dose-dependently attenuated OGD-induced cell injury. The expression levels of HIF-1α, BNIP3, and the Bax/Bcl-2 increased significantly with increasing OGD duration. Overexpression of HIF-1α decreased cell viability, increased BNIP3 expression, and Bax/Bcl-2 ratio; siRNA-HIF-1α showed the opposite effect. TMP treatment suppressed HIF-1α, BNIP3 expression, and the Bax/Bcl-2 ratio and was reversed by HIF-1α overexpression. Our study shows that TMP protects OGD-damaged PC12 cells by inhibiting the HIF-1α/BNIP3 pathway, which provides new insights into the mechanism of TMP and its neuroprotective potential.

Gomisin N attenuated cerebral ischemia-reperfusion injury through inhibition of autophagy by activating the PI3K/AKT/mTOR pathway

BackgroundIschemic stroke is a major global cause of mortality and permanent disability. Studies have shown that autophagy is essential to maintain cell homeostasis and inevitably lead to neuronal damage after cerebral ischemia. Gomisin N (GN), lignin isolated from Schisandra chinensis, possesses multiple pharmacological activities. However, there is no research on the potential of GN for neuroprotection in ischemic stroke. PurposeThe current work aimed to explore the potential therapeutic possibilities of GN on ischemic stroke and investigate the underlying molecular mechanisms. Study DesignThe neuroprotective effects of GN on PC12 cells induced by oxygen glucose deprivation/reoxygenation (OGD/R) and mice with middle cerebral artery occlusion/reperfusion (MCAO/R) injury were investigated. MethodsOn day 3 after ischemia, the infarct volume and neurological function were assessed. The level of autophagy was measured in vivo and in vitro using Transmission electron microscopy (TEM) and Monodansylcadaverine (MDC) staining. The interaction between GN and PI3K/AKT/mTOR pathway was investigated by molecular docking. Additionally, the expressions of critical proteins in the PI3K/AKT/mTOR signaling pathway and autophagy markers were determined by western blotting. ResultsIn compared to the Model group, GN might considerably improve the neurological and locomotor function following a stroke, as well as lower the volume of the cerebral infarct volume and the number of autophagosomes. GN therapy may suppress autophagy by activating the PI3K/Akt/mTOR signaling pathway in the penumbra. In vitro, MDC and TEM results showed that GN treatment obviously suppressed autophagy. Meanwhile, GN downregulated LC3II/LC3I expression ratio while upregulated the p62 expression level. In further studies, GN dramatically boosted the expression ratios of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR proteins in PC12 cells following OGD/R damage. However, the PI3K inhibitor (LY294002) reversed the increase of p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR expression ratio induced by GN administration. Also, LY294002 significantly partially attenuated GN induced reduction of autophagy and increase of cell viability compared with GN treatment alone. ConclusionsHere, we first demonstrate the neuroprotective effects of GN on MCAO mice and OGD/R induced PC12 cells injury. A possible mechanism by which GN prevents ischemic stroke is proposed: GN could restrain autophagy by stimulating the PI3K/AKT/mTOR signaling pathways. More effects and mechanisms of GN on the rehabilitation of ischemic stroke are worthy to be explored in the future.

Circ_TLK1 knockdown alleviates oxygen-glucose deprivation/reoxygenation-induced PC12 cell injury by regulating microRNA-136-5p/follistatin like-1 axis.

Circular RNAs (circRNAs) are aberrantly expressed in the central nervous system (CNS) and are involved in diverse CNS diseases. However, the functions of circRNAs in ischemic stroke (IS) are largely unknown. In this study, we aimed to explore the effect of circ_TLK1 in oxygen-glucose deprivation/reoxygenation (OGD/R)-induced PC12 cell injury. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed for the levels of circ_TLK1, TLK1, microRNA-136-5p (miR-136-5p), and follistatin like-1 (FSTL1). RNase R and Actinomycin D assays were conducted to analyze the features of circ_TLK1. 3-(4, 5-ethynyl-2'-deoxyuridine [EdU] assay and 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) assay were adopted to analyze cell proliferation capacity. Flow cytometry analysis was applied to determine cell death. Western blot assay was employed to measure protein levels. The release of lactate dehydrogenase (LDH) was measured with specific kits. The interaction between circ_TLK1 and miR-136-5p, as well as miR-136-5p and FSTL1, was verified by Dual-luciferase reporter assay. Circ_TLK1 was upregulated in OGD/R-injured PC12 cells. OGD/R treatment inhibited cell proliferation, promoted cell death, and increased LDH release in PC12 cells, while circ_TLK1 knockdown partially alleviated OGD/R-induced PC12 cell injury. Circ_TLK1 directly bound to miR-136-5p and miR-136-5p inhibition reversed the effect of circ_TLK1 knockdown on OGD/R-induced PC12 cell damage. Moreover, FSTL1 was targeted by miR-136-5p. MiR-136-5p upregulation inhibited PC12 cell injury induced by OGD/R, while FSTL1 overexpression partially reversed the effect. Circ_TLK1 knockdown ameliorated OGD/R-induced PC12 cell injury by modulating miR-136-5p and FSTL1, which might provide a new understanding of IS treatment.

Design, synthesis of amide derivatives of scutellarin and their antileukemia and neuroprotective activities

A unique series of amide-scutellarin derivatives were designed and synthesized in order to develop the function of scutellarin further. The antiproliferative activity of all target compounds against two human leukemia cell line was evaluated. Among them, compounds 6g and 7c displayed the most antitumor activities against HL-60 and THP-1 cells. Moreover, all compounds were also assayed for their neuroprotective activity against hydrogen peroxide (H2O2)-induced PC12 cell injury, and the majority of the compounds had moderate to good neuroprotective properties. These findings confirmed that these target compounds could be used as antileukemia or neuroprotective candidates in the future.

RNA-seq based transcriptome analysis of ethanol extract of saffron protective effect against corticosterone-induced PC12 cell injury

BackgroundAt present, oral antidepressants are commonly used in the clinical treatment of depression. However, the current drug treatment may lead to more serious adverse reactions. Therefore, we focus on Chinese traditional medicine, trying to find an effective and safe alternative or complementary medicine. Crocus sativus (saffron) is a traditional Chinese herbal medicine, which is typically used in the clinic to regulate anxiety, insomnia, amnesia, and other mental disorder. The study aimed to explore the neuroprotective effect of ethanol extract of saffron (EES) on corticosterone (CORT)- induced injury in PC12 cells and further explored its potential mechanism.MethodsThe authenticity of saffron and the active components of EES were identified by a water test and ultra-performance liquid chromatography-time of flight mass spectrometry system. The screening of cytotoxicity for PC12 cells was incubated with EES in different concentrations for 24 h, and the protective efficacy of EES on CORT (500 μM) -induced PC12 cell injury, cell viability was assessed by Cell Counting Kit-8 (CCK-8) assay. The differentially expressed genes (DEGs) of EES-protected PC12 cells were analyzed using the RNA-seq method, and the results were analyzed for GO and KEGG enrichment. The results of RNA-seq were verified by qPCR analysis.ResultsThe saffron was initially identified as authentic in the water test and 10 compounds were identified by Ultra Performance Liquid Chromatography (UPLC)- Mass Spectrometry (MS). The results of CCK-8 demonstrated that EES at concentrations above 640 μg/mL exerted a certain cytotoxic effect, and PC12 cells pretreated with EES (20, 40, and 80 μg/mL) significantly reversed the 500 μM CORT-induced cell death. RNA-seq analysis showed that EES regulated 246 differential genes, which were mainly enriched in the MAPK signaling pathway. Dusp5, Dusp6, Gadd45b, Gadd45G, and Pdgfc were further validated by qPCR. Experimental data showed that the results of qPCR were consistent with RNA-seq.ConclusionsThese findings provide an innovative understanding of the molecular mechanism of the protective effect of EES on PC12 cells at the molecular transcription level, and Dusp5, Dusp6, Gadd45b, Gadd45g, and Pdgfc may be potential novel targets for antidepressant treatment.

Exogenous Carbon Monoxide Produces Rapid Antidepressant- and Anxiolytic-Like Effects.

Carbon monoxide (CO), a byproduct of heme catalyzed by heme oxygenase (HO), has been reported to exert antioxidant and anti-inflammatory actions, and to produce significant neuroprotective effects. The potential effects of CO and even HO on depressive-like behaviors are still poorly understood. Utilizing several approaches including adeno-associated virus (AAV)-mediated overexpression of HO-1, systemic CO-releasing molecules (CO-RMs), CO-rich saline or CO gas treatment procedures in combination with hydrogen peroxide (H2O2)-induced PC12 cell injury model, and lipopolysaccharide (LPS)-induced depression mouse model, the present study aimed to investigate the potential antidepressant- and anxiolytic-like effects of endogenous and exogenous CO administration in vivo and in vitro. The results of in vitro experiments showed that both CO-RM-3 and CO-RM-A1 pretreatment blocked H2O2-induced cellular injuries by increasing cell survival and decreasing cell apoptosis and necrosis. Similar to the effects of CO-RM-3 and CO-RM-A1 pretreatment, AAV-mediated HO-1 overexpression in the dorsal hippocampus produced significant antidepressant-like activities in mice under normal conditions. Further investigation showed that the CO gas treatment significantly blocked LPS-induced depressive- and anxiety-like behaviors in mice. Taken together, our results suggest that the activation of HO-1 and/or exogenous CO administration produces protective effects and exerts antidepressant- and anxiolytic-like effects. These data uncover a novel function of the HO-1/CO system that appears to be a promising therapeutic target for the treatment of depression and anxiety.

Protective effect of α-asarone and β-asarone on Aβ -induced inflammatory response in PC12 cells and its.

To investigate effects of α-asarone and β-asarone on induced PC12 cell injury and related mechanisms. Aβ toxic injury cell model was induced by Aβ in PC12 cells. PC12 cells were divided into blank control group, model control group, α-asarone group (0.5, 1.0, β-asarone group (6.3, 12.5, vasoactive intestinal peptide (VIP) group, and VIP antagonist control group. Cell survival rate was detected by CCK-8 kit; cell apoptosis rate was detected by flow cytometry. The levels of inflammatory cytokines interleukin (IL)-1, , tumor necrosis factor (TNF)-α, oxidation-related inducible nitric oxide synthase (iNOS), nitric oxide (NO), apoptosis factors caspase-3 and p53 were detected by ELISA method. The expressions of C-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK) were detected by Western blotting. Compared with model control group, cell survival rates of group, β-asarone group and VIP group increased; the cell apoptosis rate decreased; levels of apoptosis-related factors caspase-3, p53, inflammatory factors IL-1, TNF-α decreased; IL-10 level increased; levels of oxidization-related factors iNOS and NO decreased; the expression of JNK and p38MAPK protein decreased (all <0.05). After VIP antagonist intervention, the survival rate of β-asarone group decreased; apoptosis rate increased; apoptosis related factors caspase-3, p53, inflammatory factors IL-1, TNF-α increased; IL-10 decreased; oxidation related factors iNOS and NO increased; the expression of JNK and p38MAPK protein increased (all <0.05); while there were no significant changes in these indicators of α-asarone group (all >0.05). α-asarone and β-asarone have protective effects on PC12 cell injury induced by Aβ. β-asarone may inhibit inflammatory factors and oxidation-related factors through promoting VIP secretion, regulating JNK/MAPK pathway, and reducing PC12 cell apoptosis; however, the effect of α-asarone may be not related to VIP secretion.

Phthalimide-(N-alkylbenzylamine) cysteamide hybrids as multifunctional agents against Alzheimer's disease: Design, synthesis, and biological evaluation.

The complex pathogenesis of Alzheimer's disease (AD) calls for multi-target approach for disease treatment. Herein, based on the MTDLs strategy, a series of phthalimide-(N-alkylbenzylamine) cysteamide hybrids were designed, synthesized, and investigated in vitro for the purpose. Most of the target compounds were found to be potential multi-target agents. In vitro results showed that compound 9e was the representative compound in this series, endowed with high EeAChE and HuAChE inhibitory potency (IC50 =1.55µm and 2.23µm, respectively), good inhibitory activity against self-induced Aβ1-42 aggregation (36.08% at 25µm), and moderate antioxidant capacity (ORAC-FL value was 0.68 Trolox equivalents). Molecular docking studies rationalized the binding mode of 9e in both PAS and CAS of AChE. Moreover, 9e displayed excellent ability to against H2 O2 -induced PC12 cell injury and penetrate BBB. Overall, these results highlighted that compound 9e was an effective and promising multi-target agent for further anti-AD drug development.

Protective effect of 1, 25(OH)_2D_3 on Aβ_(1-42)-induced pyrolysis in PC12 cells

To investigate the protective effect of 1, 25(OH)_2D_3 on Aβ_(1-42)-induced pyrolysis in PC12 cells. The Alzheimer& apos; s disease model in PC12 cells was established with 20 μmol/L Aβ_(1-42). The experiment was divided into control group, model group(20 μmol/L Aβ_(1-42)) and 1, 25(OH)_2D_3 groups(1, 10, 100 nmol/L 1, 25(OH)_2D_3+20 μmol/L Aβ_(1-42)). Cell activity was detected by CCK-8, cell membrane permeability was detected by AO/EB staining, lactic dehydrogenase(LDH)and interleukin-1β(IL-1β)were detected by colorimetry and ELISA, NOD-like receptor family protein 1(NLRP1), cysteinyl aspartate specific proteinase-1(caspase-1)and gasdermin D(GSDMD)protein expression were detected by Western Blot. Compared with the control group, the cell activitywas significantly decreased(P& lt; 0. 01), cell membrane permeability, the level of LDH and IL-1β, and the expression of NLRP1, caspase-1 and GSDMD were significantly increased(P& lt; 0. 01). Compared with the model group, the cell activity was significantly increased(P& lt; 0. 01), cell membrane damage was decreased in PC12 cells exposed to 1, 25(OH)_2D_3. The level of LDH and IL-1β were significantly decreased(P& lt; 0. 01) in PC12 cells exposed to 10 and 100 nmol/L 1, 25(OH)_2D_3. The expression of NLRP1 and GSDMD in 1 nmol/L 1, 25(OH)_2D_3 group was decreased(P& lt; 0. 05), and the decrease was more significant in 10 and 100 nmol/L 1, 25(OH)_2D_3 groups(P& lt; 0. 01). The expression of caspase-1 was significantly decreased in 10 and 100 nmol/L 1, 25(OH)_2D_3 groups(P& lt; 0. 05, P& lt; 0. 01). 1, 25(OH)_2D_3 exerts a significant protective effect against Aβ_(1-42)-induced PC12 cells injury through inhibition of neuronal pyrolysis.

LncRNA-MIAT promotes neural cell autophagy and apoptosis in ischemic stroke by up-regulating REDD1

BackgroundIschemic stroke (IS) accounts for 80% of stroke incidence, which has an impact on the life quality of patients. Long non-coding RNA (LncRNA), a class of non-coding transcripts greater than 200 nucleotidesin length, has been extensively studied in cerebrovascular diseases. Myocardial infarction associated transcript (MIAT) is highly expressed in nervous system. Therefore this study aims to explore the role of LncRNA MIAT in IS and to clarify its underlying mechanism, providing therapeutic value for the treatment of IS. MethodsThe neurological function of rats was evaluated by neurological deficit score. Triphenyltetrazolium chloride (TTC) staining was used to detect infarct area in brain tissues. Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to examine the expression of MIAT. Western blotting was used to detect the expressions of REDD1, p-mTOR, autophagy-related proteins LC3 and p62, and apoptotic-related proteins Bax, cleaved-caspase3, Bcl-2. Flow cytometry was applied to examine neuronal cell apoptosis. RNA pull-down and RIP assay was used to verify the binding of MIAT and REDD1. The level of REDD1 ubiquitination was detected by ubiquitination and Co-immunoprecipitation (Co-IP) assay. ResultsThe expressions of MIAT and REDD1 were increased in IS rats and oxygen-glucose deprivation/reoxygenation (OGD/R)-induced PC12 cell injury. After interference with si-MIAT, the results of flow cytometry showed that the rate of apoptosis was reduced. Western blotting results showed that the expression of LC3II/LC3I, Bax, and cleaved-caspase3 was decreased, while the expression of p-mTOR, p62, and Bcl-2 was increased. RNA pull-down and RIP assay found the binding relationship between MIAT and REDD1, and interference with si-MIAT down-regulated the expression of REDD1. The level of REDD1 ubiquitination was increased and the expression of REDD1 was decreased after interference with si-MIAT in PC12 cells. Co-IP results showed that interference with si-MIAT enhanced the binding ability of CUL4A-DDB1 and REDD1. ConclusionAltogether, MIAT promotes autophagy and apoptosis of neural cells and aggravates IS by up-regulating the expression of REDD1.

P53-mediated ferroptosis is required for 1-methyl-4-phenylpyridinium-induced senescence of PC12 cells

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and striatum. Aging is the most important risk factor of PD. Ferroptosis is an iron-dependent form of cell death associated with PD. However, it is not clear whether ferroptosis accelerates PD by promoting cellular senescence. This study investigated the mechanism of 1-methyl-4-phenylpyridinium (MPP+) -induced PC12 cells injury. We found that MPP+ induced cell senescence with increased β-galactosidase activity and the expression of p53, p21 and p16 activation in cells. In addition, MPP+ treatment showed smaller mitochondria and increased membrane density, downregulation of ferritin heavy chain 1 expression and upregulation of acyl-CoA synthetase long chain family member 4 expression, and enhanced levels of oxidative stress, which were important characteristics of ferroptosis. Ferrostatin-1 (Fer-1), a ferroptosis inhibitor, was tested to eliminate MPP+-induced cell senescence. Fer-1 downregulated the expression of p53 and upregulated the expression of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase-4 (GPX4) in MPP+-induced ferroptosis. Inhibition of p53 eliminated cell senescence by upregulation the expression of of SLC7A11 and GPX4. Thus, these results suggest that MPP+ induces senescence in PC12 cells via the p53/ SLC7A11/ GPX4 signaling pathway in the ferroptosis regulation mechanism.

Protosappanin A Maintains Neuronal Mitochondrial Homeostasis through Promoting Autophagic Degradation of Bax.

Cerebral ischemia is accompanied by mitochondrial integrity destruction. Thus, reversion of mitochondrial damage holds great potential for cerebral ischemia therapy. As a crucial Bcl-2 family member, pro-apoptotic Bax protein is a main effector of mitochondrial permeabilization and plays an important role in mitochondrial homeostasis. However, there is still a lack of an effective cerebral protective strategy through selectively targeting Bax. In this study, we reported that natural small-molecule protosappanin A (PTA) showed a significant mitochondrial protective effect on oxygen-glucose deprivation/reperfusion (OGD/R)-induced PC12 cells injury through increasing ATP production and maintaining mitochondrial DNA (mtDNA) content. The mechanism study revealed that PTA selectively induced pro-apoptotic protein Bax degradation, without affecting other Bcl-2 family members such as Bcl-2, Bcl-xl, Bad, Puma, Bid, Bim, and Bik. In addition, we found that PTA promoted the association of autophagosomal marker LC3B to Bax for its degradation via an autophagy-dependent manner but not the ubiquitin-proteasome pathway. Collectively, our findings offered a new pharmacological strategy for maintaining mitochondrial function by inducing autophagic degradation of Bax and also provided a novel drug candidate against ischemic neuronal injury.

Neuroprotective compounds from the resinous heartwood of Aquilaria sinensis

Six undescribed compounds, including three sesquiterpenoids [(4S,5S,7S,8S,11R)-7-hydroxyguai-1(10)-en-8,12-olide, aquilarisinone, and 2Z,7(13),9E-humulatrien-12-ol-5-one], one diphenylpentanone [1-(2-hydroxyphenyl)-5-phenylpentan-3-one], and two 2-(2-phenylethyl)chromones (6-epiagarotetrol and triepoxyhexahydrochromone A), along with 15 known compounds, were isolated from the resinous heartwood of Aquilaria sinensis (Thymelaeaceae). Their structures were determined by mass (MS) and nuclear magnetic resonance (NMR) spectroscopic data. The absolute configuration of (4S,5S,7S,8S,11R)-7-hydroxyguai-1(10)-en-8,12-olide was confirmed by X-ray diffraction analysis, and the configurations of (4S,7S,8S,10R,11R)-7,10-epoxyguai-1(5)-en-8,12-olide, aquilarisinone, 6-epiagarotetrol, and triepoxyhexahydrochromone A were confirmed by electronic circular dichroism (ECD) calculations. The neuroprotective activities of the compounds were evaluated using models of BACE1 inhibition and PC12 cells with corticosterone- and 1-methyl-4-phenylpyridine ion (MPP+)-induced damage. At concentrations of 1, 2, and 5 μM, triepoxyhexahydrochromone A, (+)-(7R,10R)-selina-4,11(13)-diene-12,15-dial, (−)-(5R,7R,10R)-12,15-dioxo-α-selinene, and (+)-(1R,4S,5R)-1β-hydroxyeremophila-7(11),9-dien-8-one exerted significant protective effects (p < 0.01) on PC12 cell injury induced by corticosterone, while triepoxyhexahydrochromone A and (−)-(5R,7R,10R)-12,15-dioxo-α-selinene exerted significant protective effects (p < 0.01) on MPP+-induced PC12 cell injury at concentrations of 1, 2, and 5 μM. No compounds produced significant inhibitory effects on BACE1, with inhibition rates of less than 20% observed at a concentration of 20 μM.

Sulfur dioxide derivatives produce antidepressant- and anxiolytic-like effects in mice

Sulfur dioxide (SO2) can be endogenously generated from sulfur-containing amino acids in animals and humans. Increasing evidence shows that endogenous SO2 may act as a gaseous molecule to participate in many physiological and pathological processes. However, the role of SO2 and its derivatives in the central nervous system remains poorly understood. The present study explored the protective effects of exogenous SO2 derivatives (Na2SO3:NaHSO3, 3:1 M/M) on cellular injury in vitro by using the cell proliferation assay (MTS), cell counting kit 8 assay (CCK-8), and cyto-flow assay in the corticosterone (CORT)-induced PC12 cell injury model. We also examined the antidepressant and anxiolytic effects of SO2 derivatives on the chronic mild stress (CMS)-induced depression mouse model by using the open field test, novelty suppressed feeding test, forced swimming test, tail suspension test, and sucrose preference test. In the MTS and CCK-8 assays, we found that preexposure of SO2 derivatives significantly blocked CORT-induced decrease of cellular survival without causing any negative effects. Results from the cyto-flow assay indicated that treatment with SO2 derivatives could reverse CORT-induced early and late apoptosis of PC12 cells. Systemic treatment with SO2 derivatives produced markedly antidepressant- and anxiolytic-like activities in mice under normal condition and rapidly reversed CMS-induced depressive- and anxiety-like behaviors. In conclusion, these findings indicate that exogenous SO2 derivatives show protective properties against the detrimental effects of stress and exert antidepressant- and anxiolytic-like actions. The present study suggests that exogenous SO2 derivatives are potential therapeutic agents for the treatment of depression, anxiety, and other stress-related diseases.