Ischemia/reperfusion (I/R) injury is one of the major causes of cardiovascular disease. Gypenoside A (GP), the main active component of Gynostemma pentaphyllum, alleviates myocardial I/R injury. Circular RNAs (circRNAs) and microRNAs (miRNAs) are involved in the I/R injury. We explored the protective effect of GP on human cardiomyocytes (HCMs) via the circ_0010729/miR-370-3p/RUNX1 axis. Overexpression of circ_0010729 abolished the effects of GP on HMC, such as suppression of apoptosis and increase in cell viability and proliferation. Overexpression of miR-370-3p reversed the effect of circ_0010729 overexpression, resulting in the stimulation of HMC viability and proliferation and inhibition of apoptosis. The knockdown of miR-370-3p suppressed the effects of GP in HCMs. RUNX1 silencing counteracted the effect of miR-370-3p knockdown and maintained GP-induced suppression of apoptosis and stimulation of HMC viability and proliferation. The levels of RUNX1 mRNA and protein were reduced in cells expressing miR-370-3p. In conclusion, this study confirmed that GP alleviated the I/R injury of myocardial cell via the circ_0010729/miR-370-3p/RUNX1 axis.

Searching for new anti-ischemic stroke (anti-IS) drugs has always been a hot topic in the pharmaceutical industry. Natural products are an important source of discovering anti-IS drugs. The aim of the present study is to extract, rapidly prepare and explore the neuroprotective effect of texasin, a main active constituent from Caragana jubata (Pall.) Poir., which is a kind of Tibetan medicine with a clear anti-IS effect. The results showed that 95% ethanol was the optimal extraction solvent. A three-step rapid preparation method for texasin was successfully established, with a purity of 99.2%. Texasin at the concentration of 25-100 µM had no effect on the viability of normal cultured PC12 cells; 12.5 and 25 µM texasin could enhance the viability of PC12 cells damaged by oxygen and glucose deprivation/reoxygenation (OGD/R), and their effects are comparable to the positive drug edaravone at the concentration of 50 µM. Compared with the normal group, the expression of Bcl-2 protein in OGD/R-injured PC12 cells was downregulated (p < 0.01), and that of PERK, eIF2α, ATF4, CHOP, Bax and Cleaved caspase-3 proteins were upregulated (p < 0.01, p < 0.001). Compared with the OGD/R group, 25 µM texasin could upregulate the expression of Bcl-2 protein (p < 0.01), and downregulate that of PERK, eIF2α, ATF4, CHOP, Bax and Cleaved caspase-3 proteins (p < 0.01, p < 0.001). The 7-OH and 1-O of texasin formed H-bonds with residues Cys891 of the hinge β-strand of PERK, which is crucial for kinase inhibitors. The above results suggest that the method established in the present study achieved rapid preparation of high-purity texasin. Texasin might inhibit neuronal apoptosis via the regulation of endoplasmic reticulum stress PERK/eIF2α/ATF4/CHOP signalling pathway to exert a protective effect on OGD/R-injured PC12 cells. Aiding by molecular docking, texasin was assumed to be a potential PERK inhibitor.

OBJECTIVE: It is previously reported that aldehyde dehydrogenase 2 family member (ALDH2) shows neuroprotective effects in cerebral ischemia/reperfusion injury. However, whether the protective effects are through mediating the programmed cell death is yet to be fully elucidated.
METHODS: In vitro oxygen-glucose deprivation/reoxygenation (OGD/R) model was established in HT22 cells and mouse cortical neurons. Subsequently, ALDH2 expression were assessed by qRT-PCR and western blot. The methylation status was examined by methylation-specific PCR (MS-PCR). Then, ALDH2 expression was promoted and suppressed to explore the role of ALDH2 in OGD/R-treated cells. CCK-8 assay was applied to detect cell viability, and flow cytometry was applied to evaluate cell apoptosis. Western blot was applied to detect the apoptosis-related proteins (Caspase 3, Bcl-2 and Bax), necroptosis-related proteins (RIP3 and MLKL), pyroptosis-related proteins (NLRP3 and GSDMD), ferroptosis-related protein (ACSL4 and GPX4), and autophagy-related proteins (LC3B, and p62). IL-1β and IL-18 production was evaluated by ELISA assay. Reactive oxygen species production and Fe2+ content were evaluated by the corresponding detection kit.
RESULTS: In OGD/R-treated cells, ALDH2 expression was decreased, which was due to the hypermethylation of ALDH2 in the promoter region. ALDH2 overexpression improved cell viability and ALDH2 knockdown suppressed cell viability in OGD/R-treated cells. We also found that ALDH2 overexpression attenuated OGD/R-induced cell apoptosis, pyroptosis, ferroptosis and autophagy, while ALDH2 knockdown facilitated the OGD/R-induced cell apoptosis, pyroptosis, ferroptosis and autophagy.
CONCLUSIONS: Collectively, our results implied that ALDH2 attenuated OGD/R-induced cell apoptosis, pyroptosis, ferroptosis and autophagy to promote cell viability in HT22 cells and mouse cortical neurons.

Severe cerebral ischemia/reperfusion injury has been shown to induce high-level autophagy and neuronal death. Therefore, it is extremely important to search for a target that inhibits autophagy activation. Long non-coding RNA MEG3 participates in autophagy. However, it remains unclear whether it can be targeted to regulate cerebral ischemia/reperfusion injury. Our results revealed that in oxygen and glucose deprivation/reoxygenation-treated HT22 cells, MEG3 expression was obviously upregulated, and autophagy was increased, while knockdown of MEG3 expression greatly reduced autophagy. Furthermore, MEG3 bound miR-181c-5p and inhibited its expression, while miR-181c-5p bound to autophagy-related gene ATG7 and inhibited its expression. Further experiments revealed that mir-181c-5p overexpression reversed the effect of MEG3 on autophagy and ATG7 expression in HT22 cells subjected to oxygen and glucose deprivation/reoxygenation. In vivo experiments revealed that MEG3 knockdown suppressed autophagy, infarct volume and behavioral deficits in cerebral ischemia/reperfusion mice. These findings suggest that MEG3 knockdown inhibited autophagy and alleviated cerebral ischemia/reperfusion injury through the miR-181c-5p/ATG7 signaling pathway. Therefore, MEG3 can be considered as an intervention target for the treatment of cerebral ischemia/reperfusion injury. This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University, China (approval No. XF20190538) on January 4, 2019.

UNASSIGNED: The roles of long non-coding RNA (lncRNAs) in ischemic stroke (IS) have been widely illustrated. Here, we focused on the function and mechanism of lncRNA SNHG7 in IS.
UNASSIGNED: Middle cerebral artery occlusion (MCAO) was used for inducing mice to establish IS models in vivo. Oxygen and glucose deprivation/reoxygenation (OGD/R) was used for treating PC12 cells to establish IS models in vitro. Relative expression of SNHG7 and miR-9 was determined by qRT-PCR. The neuronal injury was assessed by measuring relative activity of ROS, malondialdehyde (MDA) level and cell viability. Cell viability was determined by MTT assay. Dual-luciferase reporter (DLR) assay was employed to test the target of SNHG7 or miR-9. Western blot was used to determine the protein expression of SIRT1. Apoptosis rate was measured by flow cytometry.
UNASSIGNED: SNHG7 was down-regulated and miR-9 was up-regulated by MCAO treatment in brain tissues of mice and by OGD/R treatment in PC12 cells. Overexpression of SNHG7 or suppression of miR-9 decreased the relative activity of ROS and the MDA level as well as enhancing cell viability, and SNHG7 reduced apoptosis rate in OGD/R-induced PC12 cells (IS cells). MiR-9 was targeted by SNHG7 and SIRT1 was targeted by miR-9. The protein expression of SIRT1 was reduced by OGD/R treatment in PC12 cells. The suppressive effects of SNHG7 on the relative activity of ROS, the MDA level and apoptosis rate as well as the promotion effect of SNHG7 on cell viability were reversed by miR-9 mimics or sh-SIRT1 in IS cells.
UNASSIGNED: LncRNA SNHG7 alleviated OGD/R-induced neuronal injury by mediating miR-9/SIRT1 axis in vitro.

This study is designed to explore the role of miR-485-5p in hypoxia/reoxygenation-induced neuronal injury in primary rat cortical neurons. Hypoxia/reoxygenation model was established through oxygen and glucose deprivation/reoxygenation (OGD/R). RN-c cells were transfected with miR-485-5p mimics, miR-485-5p inhibitors, si-SOX6, pCNDA3.1-SOX6 or miR-485-5p + pCDNA3.1-SOX6, in which cell viability, apoptosis, lactate dehydrogenase (LDH) release rate were assessed. Western blot detected the protein expressions of apoptotic-related proteins (caspase3, Bcl-2, Bax) and the phosphorylated level of ERK1/2. The potential binding sites between miR-485-5p and SOX6 were predicted by STARBASE and identified using dual luciferase reporter gene assay. OGD/R-treated RN-c cell presented increases in apoptosis and LDH release rate as well as a decrease in cell viability. miR-485-5p was downregulated while SOX6 was upregulated in OGD/R-treated RN-c cells. Overexpression of miR-485-5p or SOX6 knockdown rescued cell viability and Bcl-2 expression, while attenuated apoptosis, LDH release rate, expression of SOX6 and the phosphorylated level of ERK1/2. Consistently, miR-485-5p inhibition led to the reverse pattern. Co-transfection of miR-485-5p and SOX6 reversed the protective effect of miR-485-5p on OGD/R-induced neuronal apoptosis. miR-485-5p can directly target SOX6. Together, miR-485-5p inhibited SOX6 to alleviate OGD/R-induced apoptosis. Collectively, miR-485-5p protects primary cortical neurons against hypoxia injury through downregulating SOX6 and inhibiting MAPK pathway.

Recent studies have indicated that resveratrol has protective effects against cerebral ischemia/reperfusion injury. However, the best therapeutic time for resveratrol treatment after acute ischemic stroke remains unknown. We aim to investigate whether resveratrol, administrated at different times after neuronal oxygen and glucose deprivation/reoxygenation (OGD/R) reduced neuronal injury in vitro. There were six experimental groups: normal, model, resveratrol pretreatment, resveratrol post-treatment, resveratrol OGD-treatment, and resveratrol whole-processing group. We found that resveratrol in a concentration-dependent manner decreased the activity of lactate dehydrogenase (LDH) and increased the activity of superoxide dismutase (SOD). Moreover, resveratrol, administrated at different times, increased neuronal viability, reduced neuronal apoptosis, upregulated the protein expressions of Nuclear factor erythroid 2-related factor 2 (Nrf-2), NAD(P)H: quinone oxidoreductase 1 (NQO-1), heme oxygenase 1 (HO-1), and Bcl-2, downregulated the protein expression of Caspase-3, and promoted Nrf-2 to transfer into the nuclei from the cytoplasm. The most effective treatment group was the whole-processing treatment group. These results suggest that resveratrol treatment at different times increased neuronal viability and inhibited neuronal apoptosis in vitro, at least in part, via enhancing the activation of the Nrf-2 signaling pathway.

In this study, we provided evidence that curcumin could be a promising therapeutic agent for ischemic stroke by activating neuroprotective signaling pathways. Post oxygen and glucose deprivation/reoxygenation (OGD/R), primary mouse cortical neurons treated with curcumin exhibited a significant decrease in cell death, LDH release and enzyme caspase-3 activity under OGD/R circumstances, which were abolished by flotillin-1 downregulation or extracellular signal-regulated kinase (ERK) inhibitor. Moreover, flotillin-1 knockdown led to suppression of curcumin-mediated ERK phosphorylation under OGD/R condition. Based on these findings, we concluded that curcumin could confer neuroprotection against OGD/R injury through a novel flotillin-1 and ERK1/2 pathway.

Hydrogen peroxide (H2O2) plays an important role in pathological conditions, such as cerebral ischemia-reperfusion (I-R) injury. Fluorescent probes may serve as valuable tools to detect the amount, temporal and spatial distribution of H2O2 in living cells. To investigate the role of lysosomal H2O2 involved in cerebral I-R injury, we designed and synthesized a lysosome-targetable two-photon fluorescent probe ztl-4, through expansion and substitution of the original pyridazinone scaffold, conjugation of electronic-donating aromatic ring and precise terminal modification of the alkyl linker. The probe ztl-4 exhibited fast, sensitive and highly selective response toward H2O2. ztl-4 could image exogenous H2O2 in SH-SY5Y cells and brain slices. In addition, ztl-4 was located in lysosomes with high colocalization coefficient compared with LysoTracker. ztl-4 was further applied for detecting the endogenous generation of H2O2 in SH-SY5Y cells subjected to oxygen and glucose deprivation (OGD) or OGD/reoxygenation (OGD/R) injury. Both OGD- and OGD/R-induced cell injury caused a time-dependent increase of H2O2 production within lysosomes. Moreover, OGD/R-treated cells showed much more amount of H2O2 than OGD-treated cells, indicating that reoxygenation will promote H2O2 accumulation in lysosomes of post-hypoxia cells. Therefore, the probe is suitable for monitoring the dynamic changes of lysosomal H2O2 in cells.