MiR-582-5p attenuates neonatal hypoxic-ischemic encephalopathy by targeting high mobility group box 1 (HMGB1) through inhibiting neuroinflammation and oxidative stress

Background: MiR-582-5p has been demonstrated to protect against ischemic stroke. However, its implication in the progression of neonatal hypoxic-ischemic encephalopathy (HIE) has not been explored. Methods: In this study, we used an in vitro model of oxygen-glucose deprivation (OGD) to investigate the protective effect of miR-582-5p on PC12 cells. OGD-induced inhibition of cell viability and promotion of cell death was assessed by CCK-8 assay and flow cytometry. Real-time PCR and enzyme-linked immunosorbent assay (ELISA) were utilized to examine the levels of inflammatory cytokines. The effects of miR-582-5p on OGD-induced oxidative injury were assessed by the determination of oxidative stress indicators. Furthermore, dual-luciferase reporter assay and gain-offunction assay were used to determine the mechanism of miR-582-5p in OGD-induced cell injury. Results : The expression of miR-582-5p was reduced upon OGD treatment in PC12 cells. Overexpression of miR-582-5p inhibited OGD-induced PC12 cell injury by regulating cell viability, apoptosis, inflammatory responses, and oxidative stress. MiR-582-5p targeted and negatively regulated high mobility group box 1 (HMGB1). MiR-582-5p presented protective effects on OGD-induced PC12 cell injury by targeting HMGB1. Conclusion: Our results indicated that miR-582-5p ameliorates neuronal injury by inhibiting apoptosis, inflammation, and oxidative stress through targeting HMGB1.

Adipose Mesenchymal Stem Cell-Derived Exosomes Enhance PC12 Cell Function through the Activation of the PI3K/AKT Pathway

Transplantation of mesenchymal stem cells has been considered as an auspicious treatment for repairing nerve injuries. The rat adrenal pheochromocytoma cell line (PC12) is one of the traditional models for the study of neuronal differentiation and neuroregeneration in vitro. However, the effects of adipose mesenchymal stem cell-derived exosomes (ADSC-exo) on PC12 cells remain unclear and to be elucidated. In our study, the effects of ADSC-exo on PC12 cells were investigated. ADSC-exo were isolated by ultracentrifugation and characterized by transmission electron microscopy, flow nanoanalysis, and western blot. The effects of ADSC-exo on PC12 cell proliferation, migration, apoptosis, and the protein levels were analyzed using CCK-8 assay and EdU incorporation assay, transwell migration assay and scratch wound assay, flow cytometry, and western blot, respectively. We successfully isolated and purified exosomes from ADSC supernatant and found that ADSC-exo treatment significantly promoted PC12 cell proliferation and migration, inhibited their apoptosis, and activated the PI3K/AKT pathway, while PI3K/AKT signaling repression using LY294002 exhibited the opposite effects. The results showed that ADSC-exo promoted proliferation and migration and inhibited apoptosis of PC12 through the activation of the PI3K/AKT pathway. Thus, the effect of ADSC-exo on PC12 cells may suggest ADSC-exo may be a promising therapeutic for nerve damage.

RNA-Seq Based Transcriptome Analysis of Ethanol Extract of Saffron Protective Effect Against Corticosterone-Induced PC12 Cell Injury

Background Saffron is a traditional Chinese herbal medicine, which is typically used in clinical to regulate anxiety, tension, and other depression-related conditions. 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. Methods The 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 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. Results The saffron was initially identified as authentic in the water test and 10 compounds were identified by UPLC-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. Conclusions These findings provide an innovative understanding of the molecular mechanism of the protective effect of EES on PC12 cells at the molecular transcription level, and the above molecules may be potential novel targets for antidepressant treatment.

Protective Effects and Mechanisms of Dendrobium nobile Lindl. Alkaloids on PC12 Cell Damage Induced by Aβ25-35

Background. Aβ deposition abnormally in the mitochondria can damage the mitochondrial respiratory chain and activate the mitochondrial-mediated apoptosis pathway, resulting in AD-like symptoms. Objective. To observe the protective effects of Dendrobium nobile Lindl. alkaloids (DNLA) on Aβ25-35-induced oxidative stress and apoptosis in PC12 cells explore its possible protective mechanisms. Methods. PC12 cells were treated with DNLA with different concentrations (0.035 mg/L, 0.3 mg/L, and 3.5 mg/L) for 6 h, followed by administration with Aβ25-35 (10 μM) for 24 h. MTT assay and flow cytometer observe the effect of DNLA on Aβ25-35-induced cytotoxicity and apoptosis of PC12 cell. Based on the mitochondrial apoptosis pathway to study the antiapoptotic effect of DNLA on this model and its relationship with oxidative stress, flow cytometer detected the level of reactive oxygen species (ROS), and ELISA kits were used to detect superoxide dismutase activity (SOD) and glutathione (GSH) content in cells. The JC-1 fluorescent staining observed the effect of DNLA on the mitochondrial membrane potential (MMP) with inverted immunofluorescence microscopy. Western blot was used to detect the levels of mitochondrial apoptosis pathway-related protein and its major downstream proteins Bax, Bcl-2, cleaved-caspase-9, and cleaved-caspase-3. Results. DNLA can significantly improve the viability and apoptosis rate of PC12 cell damage induced by Aβ25-35. It also can restore the reduced intracellular ROS content and MMP, while SOD activity and GSH content increase significantly. The expression of apoptosis-related protein Bax, cleaved-caspase-9, and cleaved-caspase-3 decreased when the Bcl-2 protein expression was significantly increased. Conclusion. These findings suggest that it can significantly inhibit the apoptosis of PC12 cell damage induced by Aβ25-35. The mechanism may reduce the level of cellular oxidative stress and thus inhibit the mitochondrial-mediated apoptosis pathway.