Current Neurovascular Research - Volume 19, Issue 1, 2022

Objective: Evidences demonstrate that propofol attenuates neuro-inflammation following brain ischemia. Moreover, LncRNA-MEG3 has been identified as an independent prognostic marker for ischemic stroke patients, and found to correlate to cerebral ischemia in animal models. Therefore, the current study explored the role of propofol in lipopolysaccharide (LPS)-mediated inflammation in cultured astrocytes, along with the molecular mechanism involved in LncRNAMEG3/ NF-ΚB axis. Methods: The primary cultured astrocytes isolated from rats were used to establish an inflammatory model, which were treated with LPS. Propofol was administrated to the primary cultured astrocytes during LPS treatment. The effects of propofol on pro-inflammatory cytokines and the LncRNAMEG3/ NF-ΚB pathway were detected by ELISA, qRT-PCR and Western Blot assay, respectively. Then, dual-luciferase assay, chromatin immunoprecipitation and RNA immunoprecipitation were used to determine the interaction between LncRNA-MEG3 and NF-ΚB. Results: Our study found propofol to significantly reduce LncRNA-MEG3 expression, which was elevated in LPS-stimulated astrocytes. Moreover, both propofol and LncRNA-MEG3 knockdown remarkably alleviated LPS-induced cytotoxicity by suppressing expressions and release of proinflammatory cytokines. Loss of LncRNA-MEG3 notably suppressed the NF-ΚB activity and its phosphorylated activation. Additionally, it was also observed that LncRNA-MEG3 could bind nuclear p65/p50, and promote the binding of NF-ΚB to IL-6 and TNF-α promoters in the nucleus, subsequently stimulating the production of inflammatory cytokines in LPS-treated astrocytes. Furthermore, a specific inhibitor of NF-ΚB, PDTC, rescued astrocytes from LPS exposure without affecting the LncRNA-MEG3 expression. Conclusion: These findings demonstrate that LncRNA-MEG3 acts as a positive regulator of NF-ΚB, mediating the neuroprotection of propofol in LPS-triggered astrocytes injury.

Background: Parkinson’s disease (PD) is associated with coiled-coil-helix-coiled-coilhelix domain containing 2 (CHCHD2) downregulation, which has been linked to reduced cyclocytase activity and increased levels of oxygen free radicals, leading to mitochondrial fragmentation and apoptosis. Little is known about how CHCHD2 normally functions in the cell and, therefore, how its downregulation may contribute to PD. Objective: This study aimed to identify such target genes using chromatin immunoprecipitation sequencing from SH-SY5Y human neuroblastoma cells treated with neurotoxin 1-methyl-4- phenylpyridinium (MPP+) as a PD model. Methods: In this study, we established a MPP+ -related SH-SY5Y cell model and evaluated the effects of CHCHD2 overexpression on cell proliferation and apoptosis. At the same time, we used high-throughput chromatin immunoprecipitation sequencing to identify its downstream target gene in SH-SY5Y cells. In addition, we verified the possible downstream target genes and discussed their mechanisms. Results: The expression level of α-synuclein increased in SH-SY5Y cells treated with MPP+, while the protein expression level of CHCHD2 decreased significantly, especially after 24 h of treatment. Chip-IP results showed that CHCHD2 might regulate potential target genes such as HDX, ACP1, RAVER2, C1orf229, RN7SL130, GNPTG, erythroid 2 Like 2 (NFE2L2), required for cell differentiation 1 homologue (RQCD1), solute carrier family 5 member 7 (SLA5A7), and NAcetyltransferase 8 Like (NAT8L). NFE2L2 and RQCD1 were validated as targets using PCR and western blotting of immunoprecipitates, and these two genes together with SLA5A7 and NAT8L were upregulated in SH-SY5Y cells overexpressing CHCHD2. Downregulation of CHCHD2 may contribute to PD by leading to inadequate expression of NFE2L2 and RQCD1 as well as, potentially, SLA5A7 and NAT8L. Conclusion: Our results suggest that CHCHD2 plays a protective role by maintaining mitochondrial homeostasis and promoting proliferation in neurons. In this study, the changes of CHCHD2 and downstream target genes such as NFE2L2/RQCD1 may have potential application prospects in the future. These findings provide leads to explore PD pathogenesis and potential treatments.

Background: Receptor-interacting serine-threonine protein kinase 3 (RIP3) was previously discovered to be an important medium in the occurrence and development of major atherosclerotic cerebral infarction. However, the role of RIP3 in acute ischemic stroke remains unclear. Objective: This study aimed to explore the correlation between plasma levels of RIP3 and acute ischemic stroke with large-artery atherosclerosis (LAA). Methods: This prospective study enrolled 116 patients with LAA, 40 healthy controls, and 30 acute ischemic stroke patients with small-artery occlusion. The patients with LAA were divided according to the quartile of plasma levels of RIP3. A logistic regression model was used for comparison. The ROC curve was performed to evaluate the predictive value. Results: In patients with LAA, the RIP3 levels in patients with poor outcomes as well as neurological deterioration were significantly higher than those with good outcomes (P < 0.001) and without neurological deterioration (P = 0.014). Patients in the highest levels of plasma RIP3 quartile were more likely to have neurological deterioration (OR, 11.07; 95% CI, 1.990-61.582) and poor outcomes (OR, 35.970; 95% CI, 5.392-239.980) compared with the lowest. The optimal cut-off value for neurological deterioration was 1127.75 pg/mL (specificity, 66.7%; sensitivity, 69.2%) and that for poor prognosis was 1181.82 pg/mL (specificity, 89.7%; sensitivity, 62.1%). Conclusion: Elevated levels of plasma RIP3 were significantly associated with neurological deterioration and poor prognosis in patients with LAA. A significant increase in plasma RIP3 levels can predict neurological deterioration and the poor prognosis of these patients.

Background: Sepsis frequently occurs in patients after infection and is highly associated with death. Septic encephalopathy is characterized by dysfunction of the central nervous system, of which the root cause is a systemic inflammatory response. Sepsis-associated encephalopathy is a severe disease that frequently occurs in children, resulting in high morbidity and mortality. Objectives: In the present study, we aimed to investigate the neuroprotective mechanism of ginsenoside Rg1 in response to septic encephalopathy. Methods: Effects of ginsenoside Rg1 on septic encephalopathy were determined by cell viability, cytotoxicity, ROS responses, apoptosis assays, and histological examination of the brain. Inflammatory activities were evaluated by expression levels of IL-1β, IL-6, IL-10, TNF-α, and MCP-1 using qPCR and ELISA. Activities of signaling pathways in inflammation were estimated by the production of p-Erk1/2/Erk1/2, p-JNK/JNK, p-p38/p38, p-p65/p65, and p-IkBα/IkBα using western blot. Results: LPS simulation resulted in a significant increase in cytotoxicity, ROS responses, and apoptosis and a significant decrease in cell viability in CTX TNA2 cells, as well as brain damage in rats. Moreover, the production of IL-1β, IL-6, IL-10, TNF-α, and MCP-1 was reported to be significantly stimulated in CTX TNA2 cells and the brain, confirming the establishment of in vitro and in vivo models of septic encephalopathy. The damage and inflammatory responses induced by LPS were significantly decreased by treatment with Rg1. Western blot analyses indicated that Rg1 significantly decreased the production of p-Erk1/2/Erk1/2, p-JNK/JNK, p-p38/p38, p-p65/p65, and p- IkBα/IkBα in LPS-induced CTX TNA2 cells and brain. Conclusion: These findings suggested that Rg1 inhibited the activation of NF-ΚB and MAPK signaling pathways, which activate the production of proinflammatory cytokines and chemokines. The findings of this study suggested that ginsenoside Rg1 is a candidate treatment for septic encephalopathy.

Background: The neuronal death upon cerebral ischemia shares not only characteristics of necrosis, apoptosis, and autophagy but also exhibits biochemical and morphological characteristics of ferroptosis. Ferroptosis is a regulated form of cell death that is considered to be an oxidative iron-dependent process. It is now commonly accepted that iron and free radicals are considered to cause lipid peroxidation as well as the oxidation of proteins and nucleic acids, leading to increased membrane and enzymatic dysfunction and finally contributing to cell death. Although ferroptosis was first described in cancer cells, emerging evidence now links mechanisms of ferroptosis to many different diseases, including cerebral ischemia. Methods: The objective of this study was to identify the key players and underlying biochemical pathways of ferroptosis, leading to cell death upon focal cerebral ischemia in mice by using immunofluorescence, Western blotting, histochemistry, and densitometry. Results: In this study, we demonstrated that cerebral ischemia induced iron-deposition, downregulated dramatically the expression of the glutathione peroxidase 4 (GPX4), decreased the expression of the nuclear receptor coactivator 4 (NCOA4), and induced inappropriate accumulation of ferritin in the ischemic brain. This supports the hypothesis that an ischemic insult may induce ferroptosis through inhibition of GPX4. Conclusion: We conclude that iron excess following cerebral ischemia leads to cell death despite activating compensatory mechanisms for iron homeostasis, as illustrated by the accumulation of ferritins. These data emphasized the presence of a cellular mechanism that allows neuronal cells to buffer iron levels.

Background: Acupuncture has been recommended as an alternative and complementary therapy for preventing and treating cerebral ischemia by the World Health Organization (WHO) for years. However, the mechanisms remain unclear. Accumulating evidence has shown that acupuncture can promote angiogenesis to attenuate brain damage after ischemic stroke. In recent years, exosome- carried microRNAs (miRNAs) activated by acupuncture have proven effective in regulating pathological changes. We, therefore, investigated whether electro-acupuncture (EA) enhanced angiogenesis in cerebral stroke via exosome-carried miR-210. Methods: We extracted and identified the exosomes from the serum of MCAO with EA treatment and injected them into MCAO rats for further observation. Simultaneously, miR-120 siRNA and HIF-1α inhibitor were transfected. Then, we evaluated the volume of infarction, pathological changes, and expression levels of angiogenic related factors of each group of rats by TTC and HE staining, transmission electron microscope (TEM), western blot, and quantitative PCR (qPCR). Results: Compared with the MCAO group, EA-Exosome (EA-EXO) treatment significantly decreased the infarct volume and the pathological damage, but miR-210 siRNA or HIF-1α inhibitor reversed the protective outcomes induced by EA-EXO. Moreover, EA-EXO treatment upregulated miR-210 and increased CD34, HIF-1α, VEGF, Notch1 protein, and mRNA expressions compared to the MCAO group. MiR-210 siRNA or HIF-1α inhibitor treatments both down-regulated those angiogenic related proteins and mRNAs. Conclusion: EA treatment could activate the HIF-1α/VEGF/Notch 1 signal pathway to facilitate angiogenesis after ischemic stroke via exosomal miR-210.

Background: β-amyloid peptides (Aβ) induced oxidative damage contributes to the pathogenesis of neurodegenerative diseases, and the cerebrovascular system is more vulnerable to oxidative stress. Our earlier study showed a clue that Genistein (Gen) might activate the Nf-E2 related factor 2 (Nrf2) pathway to protect cerebrovascular cells from oxidative damage induced by Aβ, but the specific mechanisms and regulation targets are unclear. Objective: In this study, the anti-oxidative effects and the possible targets of Gen on regulating the Nrf2 pathway in bEnd.3 cells were investigated. Cells were divided into control, Aβ25-35, Gen, and Gen+Aβ25-35 groups. Methods: Cell viability, levels of malondialdehyde (MDA), Superoxide Dismutase (SOD) activity, and nitrotyrosine were evaluated. Moreover, mRNA and/or protein expressions of Nrf2 and kelchlike ECH-associated protein 1 (Keap1) were measured. Then we transfected Keap1 over-expressed plasmid into bEnd.3 cells and measured the protein expressions of Nrf2 pathway related factors. Results: Data showed that Gen could inhibit the over-production of MDA and nitrotyrosine and activate SOD activity. Furthermore, we discovered that Gen could up-regulate Nrf2 mRNA and protein expression while down-regulating Keap1 protein expression. The Keap1 over-expressed plasmid study revealed that the up-regulation of Nrf2 protein expression induced by Gen pretreatment could be blocked by transfection of Keap1 over-expressed plasmid, and the same results were observed in Nrf2 downstream factors. Conclusion: Gen could alleviate the cerebrovascular cells' oxidative damage induced by Aβ25-35 by regulating the Nrf2 pathway, and Keap1 might be one of the targets of Gen in activating the Nrf2 pathway.

Background/Objective: Systemic immune-inflammation index (SII) is a novel inflammatory factor, which may be involved in the destruction of the blood-brain barrier (BBB) after acute ischemic stroke (AIS); however, the association between SII and symptomatic intracranial hemorrhage (sICH) in AIS patients undergoing endovascular treatment (EVT) remains unclear. Methods: Patients with acute ischemic stroke due to large vessel occlusion (AIS-LVO) who underwent EVT were consecutively enrolled. Blood samples were collected in the emergency room and SII was calculated by neutrophils × platelets/lymphocytes. Participants were categorized into tertiles according to admission SII. The main outcome was defined as the occurrence of sICH, following the European Cooperative Acute Stroke Study III (ECASS-III) criteria. Results: A total of 379 AIS-LVO patients with EVT were enrolled (median age = 71 years, 52.5% males). The median baseline National Institutes of Health Stroke Scale (NIHSS) score was 15 (IQR, 12-18). The median of SII was 820.9 × 109/L (IQR, 473.1-1345.2). Forty-three (11.3%) patients developed sICH. SII was found to be independently associated with sICH after EVT (adjusted odd ratio (OR) = 1.005 (per 10 units increase); 95% confidence interval (CI): 1.002-1.008; p = 0.002). Compared to patients in the lowest SII tertile, patients in the highest tertile had a higher risk of sICH (adj-OR 3.379; 95% CI 1.302-8.768; p = 0.012). The risk of sICH increased with the increase of SII in a dose-dependent manner (p for trend = 0.004). There was no interaction between potential modifiers and SII on sICH. Conclusion: Admission SII is positively associated with sICH in AIS-LVO patients treated with EVT. These results need to be confirmed in future studies.

Objectives: Melatonin (MT) is a pineal hormone with antineoplastic potential. This study aims to explore the therapeutic potential and mechanism of MT on glioblastoma (GBM). Methods: A human GBM cell line, LN229, was used to evaluate the function of MT. Cell viability, apoptosis, and migration were detected by CCK-8, flow cytometry, and transwell assays, respectively. The mRNA and protein expressions of specific genes were measured by qRT-PCR and western blot, respectively. The regulatory relationship between miR-16-5p and PIM1 was validated by dual luciferase reporter gene assay. A mouse xenograft model was established to prove the anti-tumor effect and related mechanisms of MT in vivo. Results: MT inhibited the viability and migration and promoted the apoptosis of LN229 cells in a dose-dependent manner. MiR-16-5p was dose-dependently up-regulated by MT in LN229 cells, negatively regulating its target PIM1. MiR-16-5p inhibitor eliminated the anti-tumor effect of MT in LN229 cells, while si-PIM1 reversed the effect of miR-16-5p inhibitor in MT-treated cells. MT inhibited the tumor growth in vivo and MT-induced PIM1 down-regulation was reversed by miR- 16-5p inhibition in tumor tissues. Conclusions: MT inhibits the malignant progression of GBM via regulating miR-16-5p-mediated PIM1.

Background: Carotid artery stenosis (CAS) patients show reduced blood flow in the ophthalmic artery. This study aimed to assess the changes in the choriocapillaris and choroidal thickness in patients with unilateral carotid artery stenosis after carotid stenting using swept-source optical coherence tomography (SS-OCT)/swept-source optical coherence tomography angiography (SSOCTA). Methods: Fifty-three mild to moderate CAS patients and 40 controls were enrolled in this study. All participants underwent digital subtraction angiography (DSA) and SS-OCT/SS-OCTAA imaging before and 4 days after carotid artery stenting. SS-OCTA was used to image and measure the perfusion of the choriocapillaris (mm2), while SS-OCT was used to image and measure the choroidal thickness (μm). The stenosed side was described as the ipsilateral eye, while the other side was the contralateral eye. Results: Choroidal thickness was significantly thinner (P = 0.024) in CAS when compared with controls. Ipsilateral eyes of CAS patients showed significantly thinner (P = 0.008) choroidal thickness when compared with contralateral eyes. Ipsilateral eyes of CAS patients showed thicker (P = 0.027) choroidal thickness after carotid artery stenting, while contralateral eyes showed thinner choroidal thickness (P = 0.039). Conclusion: Our report suggests that in vivo quantification of the choroid with the SS-OCT/SSOCTA may allow monitoring of CAS and enable the assessment of purported treatments.

Background: MicroRNAs (miRNAs) may participate in the process of vascular calcification. However, the role of microRNA-17-5p in vascular calcification has not been clarified. In this study, we showed the effects of microRNA-17-5p on vascular calcification. Materials and Methods: Vascular smooth muscle cells (VSMCs) were transfected with miR-17-5p mimics, a miR-17-5p inhibitor or negative control (NC) using Lipofectamine 2000. Then the cells were induced by an osteogenic medium. Alkaline phosphatase (ALP) activity and mineralization were determined. Osteocalcin (OC), bone morphogenetic protein 2(BMP-2), Collagen Ia (Colla), Runx2, and ankylosis protein homolog (ANKH) gene expressions were determined by reverse transcription-polymerase chain reaction. Vascular calcification was developed using a renal failure model. Results: The ALP activity was increased when miR-17-5p mimics were transfected, whereas the miR-17-5p inhibitor reduced ALP activity (p < 0.05). The number and average area of mineral nodes in the miR-17-5p mimic group was larger than those in the corresponding control and NC groups (p < 0.05). The number and average area of the mineral nodes in the miR-17-5p inhibitor group were smaller than those in the corresponding control and NC groups (p < 0.05). Bmp2, OC, Col1a and Runx2 were higher in the miR-17-5p mimics group compared to those in the control and NC groups. ANKH expression was decreased in VSMCs with the miR-17-5p mimics and increased in VSMCs with miR-17-5p inhibitor. ANKH siRNA intervention also promoted mineralization. The miR-17-5p expression was upregulated and ANKH was down-regulated in the aortic arteries with calcification. Conclusion: Our data showed that miR-17-5p may promote vascular calcification by inhibiting ANKH expression.

Background: Stroke is a serious disease that threatens human health both in China and worldwide. Identifying and establishing its risk factors are prerequisites for intervention and evaluation of prognosis. Over the years, risk factors, such as age, diabetes, and hypertension, have gradually been established. However, at present, there is no consensus on the influence of sex on the prognosis of ischaemic stroke. Aims: The aims of our research was to analyse the correlation between sex and poststroke prognosis based on the results of the Third China National Stroke Registry [CNSR-III], as well as the influence of other risk factors that may be confounded by sex on ischaemic stroke and potential interventions. Methods: The CNSR-III recruited 14146 acute ischaemic stroke [AIS] patients between 2015 and 2018. Our study included 13,972 patients who had complete follow-up information. This research analysed basic information, socioeconomic status, lifestyle habits, medical history, and poststroke prognosis. Results: There was a conspicuous relationship between sex and functional prognosis, stroke recurrence and all-cause death due to ischemic stroke in univariate analysis. Male stroke patients had a better prognosis than female patients. In multivariate analysis, we found that age, atrial fibrillation [AF], diabetes, hypertension and the severity of stroke had adverse effects on ischemic stroke prognosis. After adjustment for other risk factors, the functional prognosis of female patients at 3 months was worse than that of male patients [odds ratio [OR] 1.16, 95% confidence interval [CI], 1.025- 1.314]. Sex had a nonsignificant association with stroke recurrence at 3 months [hazard ratio [HR] 1.141, 95% CI, 0.975-1.336]. Furthermore, compared to male patients, female stroke patients had a lower cumulative death rate at 12 months [HR 0.777, 95% CI, 0.628-0.963]. Conclusion: Our study identified sex differences in stroke-related disability, recurrence, and death and attempted to explain the causes of these differences. Our study clearly showed that a large proportion of this difference could be attributed to age, socioeconomic factors, lifestyle habits, and medical history, confounded by sex differences rather than sex per se.