Sinensetin attenuates oxygen–glucose deprivation/reperfusion-induced neurotoxicity by MAPK pathway in human cerebral microvascular endothelial cells

Sinensetin is a polymethoxylated flavone with anti-inflammatory and anti-oxidative activities. This work aimed to explore the function and mechanism of sinensetin in oxygen and glucose deprivation/reperfusion (OGD/R)-induced neurotoxicity. The overlapping target genes of cerebral stroke and sinensetin were determined according to GeneCards and ParmMapper tools and were subjected to Gene Ontology (GO) annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses. Human cerebral microvascular endothelial cells (HCMECs) were stimulated with OGD/R. Neurotoxicity was investigated by Cell Counting Kit-8, lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) level, qRT-PCR, and TUNEL analysis. The proteins (p38, JNK, and ERK) in mitogen-activated protein kinase (MAPK) signaling were measured using Western blotting. Total of 50 overlapping target genes of cerebral stroke and sinensetin were predicted. Pathway analysis showed they might be involved in the MAPK pathway. Sinensetin attenuated OGD/R-induced neurotoxicity by mitigating viability reduction, LDH release, ROS generation, inflammatory response, and apoptosis in HCMECs. Sinensetin weakened OGD/R-induced activation of the MAPK pathway via decreasing the phosphorylation of p38, JNK, and ERK. The pathway inhibitors mitigated the activation of the MAPK signaling, and sinensetin exacerbated this effect. The inhibitors reversed OGD/R-induced neurotoxicity in HCMECs, and sinensetin contributed to this role. Overall, sinensetin prevents OGD/R-induced neurotoxicity through decreasing the activation of MAPK pathway.     

Circ_002664/miR-182–5p/Herpud1 pathway importantly contributes to OGD/R-induced neuronal cell apoptosis

ObjectiveApoptosis is a prominent form of neuron death in cerebral ischemia-reperfusion-induced injury. Accompanied with the pathogenesis, Circ_002664 is upregulated. However, its role in the neuron apoptosis and the underlying mechanisms are unknown.MethodsIn this study, HT22 cells were treated with oxygen glucose deprivation and reoxygenation (OGD/R). The cell viability, apoptosis, proliferation and mitochondrial potential were examined. The expressions of interested genes, Circ_002664, miR-182–5p and Herpud1, were measured. The roles of these genes in OGD/R-induced cell injury were investigated by knockdown, overexpression alone or in combination. Additionally, the interactions between Circ_002664, miR-182–5p and Herpud1 were validated by luciferase report assay. The levels of MAP2, CHOP, Cytochrome C (CYC) and cleaved caspase-3 were determined.ResultsOGD/R treatment significantly increased cell apoptosis, decreased cell proliferation and mitochondrial potential, as well as increased Circ_002664 and Herpud1 expressions, and decreased miR-182–5p level. Circ_002664 knockdown markedly inhibited the effects by OGD/R on cell survival and altered expression of miR-182–5p and Herpud1. MiR-182–5p was observed sponged by Circ_002664 and negatively mediated its effect above mentioned, and this was by directly targeting Herpud1. Additionally, it was observed that CHOP expressions were regulated by Circ_002664/miR-182–5p/Herpud1 pathway, and in turn mediated its regulation in CYC and cleaved caspase-3.ConclusionsIn summary, our data showed that the Circ_002664 importantly contributed to neuronal cell apoptosis induced by OGD/R treatment, and this might be achieved by directly targeting miR-182–5p/Herpud1 pathway.     

Endogenous level of TIGAR in brain is associated with vulnerability of neurons to ischemic injury

In previous studies,we showed that TP53-induced glycolysis and apoptosis regulator(TIGAR) protects neurons against ischemic brain injury.In the present study,we investigated the developmental changes of TIGAR level in mouse brain and the correlation of TIGAR expression with the vulnerability of neurons to ischemic injury.We found that the TIGAR level was high in the embryonic stage,dropped at birth,partially recovered in the early postnatal period,and then continued to decline to a lower level in early adult and aged mice.The TIGAR expression was higher after ischemia/reperfusion in mouse brain 8and 12 weeks after birth.Four-week-old mice had smaller infarct volumes,lower neurological scores,and lower mortality rates after ischemia than 8- and12-week-old mice.TIGAR expression also increased in response to oxygen glucose deprivation(OGD)/reoxygenation insult or H_2O_2 treatment in cultured primary neurons from different embryonic stages(E16 and E20).The neurons cultured from the early embryonic period had a greater resistance to OGD and oxidative insult.Higher TIGAR levels correlated with higher pentose phosphate pathway activity and less oxidative stress.Older mice and more mature neurons had more severe DNA and mitochondrial damage than younger mice and less mature neurons in response to ischemia/reperfusion or OGD/reoxygenation insult.Supplementation of cultured neurons with nicotinamide adenine dinuclectide phosphate(NADPH) significantly reduced ischemic injury.These results suggest that TIGAR expression changes during development and its expression level may be correlated with the vulnerability of neurons to ischemic injury.     

Plasma Levels of miR-125b-5p and miR-206 in Acute Ischemic Stroke Patients After Recanalization Treatment: A Prospective Observational Study

Introduction: Multiple microRNAs (miRNAs) participate in the response to hypoxic/ischemic and ischemia-reperfusion events. However, the expression of these miRNAs in circulation from patients with acute ischemic stroke (AIS) receiving recanalization treatment has not been examined, and whether they are associated with the severity and outcome of stroke is still unknown. Materials and methods: In this prospective cohort study, plasma levels of miR-125b-5p, miR-15a-3p, miR-15a-5p, and miR-206 were measured at 24 hours after thrombolysis with or without endovascular treatment in 94 patients with AIS, as determined by qRT-PCR. Stroke severity was assessed based on National Institutes of Health Stroke Scale (NIHSS) score and infarct lesion. Intracranial haemorrhage (ICH) was recorded. An unfavorable outcome was defined as a modified Rankin Scale score greater than 2 at day 90 after stroke. Results: miR-125b-5p and miR-206 levels were correlated with NIHSS scores (P = .014 and P = .002) and cerebral infarction volumes (P = .025 and P = .030). miR-125b-5p levels were significantly higher in patients with an unfavorable outcome than in patients with a favorable outcome (P = .002) and showed good diagnostic accuracy in discriminating the presence of an unfavorable outcome (area under the curve .735, 95% confidence interval .623-.829, P < .001). No association was found between different miRNAs and ICH. Conclusions: In AIS patients after thrombolysis with or without endovascular treatment, miR-125b-5p is a novel prognostic biomarker highly associated with an unfavorable outcome. miR-125b-5p and miR-206 levels are associated with stroke severity.     

知母须根总皂苷提取工艺的优化及其对糖氧剥夺损伤的PC12细胞保护作用研究

目的优化知母须根总皂苷提取工艺,探讨其对糖氧剥夺(oxygen-glucose deprivation,OGD)损伤的PC12细胞的保护作用。方法在单因素实验基础上,利用响应面法优化知母须根总皂苷提取工艺;建立OGD损伤的PC12细胞模型,采用终质量浓度为20、40、80 mg/L的知母须根总皂苷进行干预。分别用倒置显微镜观察细胞形态,MTT法测定细胞存活率,荧光探针二氯荧光素-双乙酸盐(DCF-DA)法测定细胞内活性氧(ROS)含量,Annexin V/PI双染法测定细胞凋亡率,蛋白免疫印记法检测凋亡信号蛋白Bcl-2、Bax表达情况。结果知母须根总皂苷提取的最优条件为乙醇体积分数72.22%,料液比1∶11,提取时间73.33 min,在此条件下,理论提取率为8.38%,实测提取率为8.33%。PC12细胞经OGD损伤4 h后细胞活力显著降低,知母须根总皂苷对其呈现保护作用,并随着知母须根总皂苷质量浓度的升高而增强;流式细胞术分析结果表明知母须根总皂苷能明显减少OGD损伤PC12细胞中ROS含量及凋亡率,免疫印迹实验结果表明知母须根总皂苷可上调PC12细胞Bcl-2表达并下调Bax表达。结论通过响应面法优化的工艺得率高、提取效果好。知母须根总皂苷对OGD损伤的PC12细胞具有一定的保护作用,其机制可能与减少OGD诱导PC12细胞ROS含量、抑制线粒体凋亡通路有关。     

雷帕霉素对PC12细胞氧糖剥夺损伤的保护作用

目的探讨雷帕霉素对PC12细胞氧糖剥夺损伤(oxygen and glucose deprivation,OGD)的作用。方法选取处于对数生长期的PC12细胞,分为正常组、OGD组、溶剂组和1 nmol、10 nmol、100 nmol、500 nmol雷帕霉素组,OGD同时给予雷帕霉素处理,8 h后通过倒置显微镜观察细胞形态学的变化、MTT法检测细胞存活率的变化以及LDH活性检测细胞受损程度。结果与正常组相比,单纯OGD组细胞活性明显下降,且具有统计学意义(P0.05);与溶剂组相比,1、10、100、500 nmol雷帕霉素组细胞存活率均存在不同程度的增高,其中10、100、500 nmol组具有统计学意义(P0.05)。结论雷帕霉素对PC12细胞OGD损伤具有保护作用。     

Enhanced SUMOylation and SENP-1 protein levels following oxygen and glucose deprivation in neurones

Here, we show that oxygen and glucose deprivation (OGD) causes increased small ubiquitin-like modifier (SUMO)-1 and SUMO-2/3 conjugation to substrate proteins in cultured hippocampal neurones. Surprisingly, the SUMO protease SENP-1, which removes SUMO from conjugated proteins, was also increased by OGD, suggesting that the neuronal response to OGD involves a complex interplay between SUMOylation and deSUMOylation. Importantly, decreasing global SUMOylation in cultured hippocampal neurones by overexpression of the catalytic domain of SENP-1 increased neuronal vulnerability to OGD-induced cell death. Taken together, these results suggest a neuroprotective role for neuronal SUMOylation after OGD.     

Persistent oxygen-glucose deprivation induces astrocytic death through two different pathways and calpain-mediated proteolysis of cytoskeletal proteins during astrocytic oncosis

Astrocytes are thought to play a role in the maintenance of homeostasis and the provision of metabolic substrates for neurons as well as the coupling of cerebral blood flow to neuronal activity. Accordingly, astrocytic death due to various types of injury can critically influence neuronal survival. The exact pathway of cell death after brain ischemia is under debate. In the present study, we used astrocytes from rat primary culture treated with persistent oxygen-glucose-deprivation (OGD) as a model of ischemia to examine the pathway of cell death and the relevant mechanisms. We observed changes in the cellular morphology, the energy metabolism of astrocytes, and the percentage of apoptosis or oncosis of the astrocytes induced by OGD. Electron microscopy revealed the co-existence of ultrastructural features in both apoptosis and oncosis in individual cells. The cellular ATP content was gradually decreased and the percentages of apoptotic and oncotic cells were increased during OGD. After 4 h of OGD, ATP depletion to less than 35% of the control was observed, and oncosis became the primary pathway for astrocytic death. Increased plasma membrane permeability due to oncosis was associated with increased calpain-mediated degradation of several cytoskeletal proteins, including paxillin, vinculin, vimentin and GFAP. Pre-treatment with the calpain inhibitor 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid (PD150606) could delay the OGD-induced astrocytic oncosis. These results suggest that there is a narrow range of ATP that determines astrocytic oncotic death induced by persistent OGD and that calpain-mediated hydrolysis of the cytoskeletal-associated proteins may contribute to astrocytes oncosis.     

Polygalasaponin F inhibits neuronal apoptosis induced by oxygen‐glucose deprivation and reoxygenation through the PI3K/Akt pathway

Cerebral ischaemia is a common cerebrovascular disease and often induces neuronal apoptosis, leading to brain damage. Polygalasaponin F (PGSF) is one of the components in Polygala japonica Houtt, and it is a triterpenoid saponin monomer. This research focused on anti‐apoptotic effect of PGSF during oxygen‐glucose deprivation and reoxygenation (OGD/R) injury in rat adrenal pheochromocytoma cells (PC12) and primary rat cortical neurons. OGD/R treatment reduced viability of PC12 cells and primary neurons. This reduced viability was prevented by PGSF, as shown by MTT assay. OGD/R insult decreased expression of Bcl‐2/Bax both in PC12 cells and primary neurons but elevated levels of caspase‐3 in primary neurons. However, PGSF may up‐regulate expression of Bcl‐2/Bax and down‐regulate caspase‐3 in these particular cells. Furthermore, Bcl‐2/Bax and the ratio between phosphorylated Akt and total Akt were decreased in PC12 cells treated with OGD/R, and both were increased by PGSF. Moreover, increase in the ratios of Bcl‐2/Bax and phosphorylated Akt/total Akt in PC12 cells was suppressed by phosphatidylinositol 3‐kinase (PI3K) inhibitor. Data suggest PGSF might prevent OGD/R‐induced injury via activation of PI3K/Akt signalling. The ability of PGSF to block the effects of OGD/R appears to involve regulation of Bcl‐2, Bax and caspase‐3, which are related to apoptosis.     

Recurrent hypoglycemia increases oxygen glucose deprivation-induced damage in hippocampal organotypic slices

More than 65% of mortality among diabetics is due to stroke and heart disease. The major side effect of intensive therapy in both type 1 and type 2 diabetics is recurrent hypoglycemic episodes (RH). Our previous study in a rat model of insulin-requiring diabetes indicated that RH exacerbates cerebral ischemic damage. Studies related to RH in hypoglycemia unawareness suggest that RH may be deleterious to outcome following cerebral ischemia owing to systemic effects, since hormonal response to hypoglycemia is impaired following RH. The goal of the present study was to determine if RH increases oxygen-glucose deprivation (OGD)-induced damage in hippocampal organotypic slices, which are devoid of systemic influence. Hippocampal slices cultured in ex vivo conditions for 9–10 days were exposed to ten 30-min episodes of “hypoglucose” (to mimic the hypoglycemic condition) medium (1.06 mM) twice a day. Slices were exposed to OGD 12 h after the last hypo/normo-glucose exposure. OGD in control slices resulted in 60% neuronal death. The percentage of cell death in RH-treated slices was significantly higher by 24% than in control slices. The results demonstrate that RH can affect brain cells in the absence of humoral influence. In conclusion, the previous exposure of hippocampal slices to RH exacerbates OGD-induced damage. Understanding the mechanism by which RH increases ischemic damage in diabetics will help improve outcome following stroke.