ARL13B promotes cell cycle through the sonic hedgehog signaling pathway to alleviate nerve damage during cerebral ischemia/reperfusion in rats.

Cerebral ischemia/reperfusion (CIRI) is a leading cause of death worldwide. A small GTPase known as ADP-ribosylation factor-like protein 13B (ARL13B) is essential in several illnesses. The role of ARL13B in CIRI remains unknown, though. A middle cerebral artery occlusion/reperfusion (MCAO/R) in rats as well as an oxygen-glucose deprivation/reoxygenation (OGD/R) models in PC12 cells were constructed. The neuroprotective effects of ARL13B against MCAO/R were evaluated using neurological scores, TTC staining, rotarod testing, H&E staining, and Nissl staining. To detect the expression of proteins associated with the SHH pathway and apoptosis, western blotting and immunofluorescence were employed. Apoptosis was detected using TUNEL assays and flow cytometry. There was increased expression of ARL13B in cerebral ischemia/reperfusion models. However, ARL13B knockdown aggravated CIRI nerve injury by inhibiting the sonic hedgehog (SHH) pathway. In addition, the use of SHH pathway agonist (SAG) can increased ARL13B expression, reverse the effects of ARL13B knockdown exacerbating CIRI nerve injury. ARL13B alleviated cerebral infarction and pathological injury and played a protective role against MCAO/R. Furthermore, ARL13B significantly increased the expression of SHH pathway-related proteins and the anti-apoptotic protein BCL-2, while decreased the expression of pro-apoptotic protein BAX, thus reducing apoptosis. The results from the OGD/R model in PC12 cells were consistent with those obtained in vivo. Surprisingly, we demonstrated that ARL13B regulates the cell cycle to protect against CIRI nerve injury. Our findings indicate that ARL13B protects against CIRI by reducing apoptosis through SHH-dependent pathway activation, and suggest that ARL13B plays a crucial role in CIRI pathogenesis.

Exosomal miR-320e through wnt2targeted inhibition of the Wnt/ß-catenin pathway allevisate cerebral small vessel disease and cognitive impairment.

BACKGROUND: Exosomal miRNAs play crucial roles in many central nervous system diseases. Cerebral small vessel disease (CVSD) is a small vessel disease that is affected by various factors. This study aimed to investigate the role of exosomal miR-320e in the Wnt/ß-catenin pathway stimulated by oxidative stress and assess its clinical correlation with psychiatric symptoms in patients with CVSD. AIM: To explore whether exosomal miR-320e could suppress the Wnt/ß-catenin pathway and play a protective role in CVSD progression, as well as examine its potential correlation with cognitive impairment and depression in patients with CVSD. METHODS: Differentially expressed exosomal miRNAs were filtered by sequencing plasma exosomes from patients with CVSD and healthy controls. Bioinformatics and dual luciferase analyses were used to confirm the binding of miR-320e to Wnt2, and the mRNA and protein levels of downstream components in the Wnt/ß-catenin pathway were evaluated when overexpressed or with knockdown of miR-320e under H2O2-induced oxidative stress. In addition, Wnt2-targeting siRNA was used to confirm the role of miR-320e in the Wnt2-mediated inhibition of the Wnt/ß-catenin pathway. A retrospective analysis was conducted among patients with CVSD to confirm the correlation between miR-320e expression and the severity of cognitive impairment and depression, which were quantified using the Montreal Cognitive Assessment (MoCA)/Executive Function Assessment (EFA), and the Hamilton Depression Scale (HAMD)/Beck Depression Inventory (BDI), respectively. RESULTS: High-throughput sequencing revealed that exosomal miR-320e was downregulated in patients with CVSD. Bioinformatics analysis and dual-luciferase reporter gene experiments showed that exosomal miR-320e inhibited the Wnt/ß-catenin pathway in response to oxidative stress by targeting the 3' noncoding region of Wnt2. Uptake of exosomes carrying miR-320e into endothelial cells could also target Wnt2 and inhibit the Wnt2/ß-catenin pathway. Elevated miR-320e expression may protect patients with CVSD from relatively severe cognitive impairment and depression, as it was found to have a positive correlation with the MoCA/EFA and HAMD/BDI scores. CONCLUSION: Our results suggest that exosomal miR-320e suppresses the Wnt/ß-catenin pathway and may play a protective role in CVSD progression.

miR-671-5p Upregulation Attenuates Blood-Brain Barrier Disruption in the Ischemia Stroke Model Via the NF-кB/MMP-9 Signaling Pathway.

Blood-brain barrier (BBB) disruption can induce further hemorrhagic transformation in ischemic stroke (IS). miR-671-5p, a micro-RNA, is abundant in the cortex of mammalian brains. Herein, we investigated the roles and potential mechanisms for the effects of miR-671-5p on BBB permeability in IS. Results showed that miR-671-5p levels were significantly downregulated in the cerebral cortex of middle cerebral artery occlusion/reperfusion (MCAO/R) C57/BL6 mice in vivo. miR-671-5p agomir administration via right intracerebroventricular injection significantly reduced infarct volume, improved neurological deficits, the axon of neurons and nerve fiber, attenuated cell injury and apoptosis, as well as reduced BBB permeability in MCAO/R mice. Treatment with miR-671-5p agomir alleviated tight junction proteins degradation, including claudin, occludin, and ZO-1 in MCAO/R mice, and these effects were reversed following NF-κB overexpression. Bend.3 brain endothelial cells were subjected to oxygen and glucose deprivation/reoxygenation (OGD/R) treatment in vivo, and then miR-671-5p agomir was transfected into the cells. This resulted in reduction of cytotoxicity, improved cell viability, trans-endothelial electrical resistance, reduced fluorescein sodium permeability, and inhibited tight junction degradation in Bend.3 OGD/R cells. However, these effects were reversed following NF-κB overexpression. These results demonstrated that upregulation of miR-671-5p in IS models in vivo and in vitro alleviated BBB permeability by targeting NF-κB/MMP-9. In summary, miR-671-5p is a potential therapeutic target for protecting BBB permeability in IS to minimize cerebral hemorrhage transformation.

Risk factors and characteristics of ischemic stroke in patients with immune thrombocytopenia: A retrospective cohort study.

OBJECTIVES: Previous research has found that patients with immune thrombocytopenia (ITP) have an increased risk of thrombosis, such as venous thromboembolism (VT), ischemic stroke (IS)/transient ischemic attack (TIA), and cardiovascular disease (CVD), but the risk factors for stroke in patients with ITP have yet to be determined. This study aims to determine the risk factors and characteristics of ischemic stroke in patients with ITP. MATERIALS AND METHODS: This study included adults with incident primary ITP diagnosed in a tertiary medical center between 2010 and 2020. The t-test and Mann-Whitney U test were used to compare the variables between IS and non-IS groups, and the multivariate logistic regression model was employed to evaluate correlations. RESULTS: The study enrolled 1824 individuals, of whom 17 (0.93%) had IS, and 138 (1:8) were randomly chosen from 1807 non-IS patients. Age was found to be substantially associated with stroke in the multivariate analysis (OR 1.07, 95% CI: 1.026-1.116; p = 0.001). We found no correlation between platelet counts (PLT) (OR 1.013, 95% CI: 0.995-1.033; p = 0.164), mean platelet volume (MPV), platelet larger cell ratio (P-LCR), prothrombin time (PT) (OR 1.455, 95% CI 0.979-2.164; p = 0.064), activated partial thromboplastin time (APTT), D-dimer, fibrinogen or antinuclear antibody (ANA) and stroke. Of 17 ITP-IS patients, 7 (53.8%) were cryptogenic, greater than the general IS population. Three (23.1%) of them had an embolic pattern. CONCLUSION: For ITP patients, age was a significant predictor of stroke. ITP-IS patients had a more cryptogenic origin, with some showing an embolic pattern.

Facilitating Mitophagy via Pink1/Parkin2 Signaling Is Essential for the Neuroprotective Effect of ß-Caryophyllene against CIR-Induced Neuronal Injury.

Mitophagy is an important mechanism for maintaining mitochondrial homeostasis through elimination of damaged or dysfunctional mitochondria following cerebral ischemia-reperfusion (CIR) injury. ß-Caryophyllene (BCP) is a natural sesquiterpene compound found in the essential oil of plants and has been shown to ameliorate CIR injury. However, whether BCP protects neurons from CIR injury by activating mitophagy is still unclear, and the underlying mechanism remains unknown. In the present study, a mouse neuron HT-22 cell of oxygen-glucose deprivation/reoxygenation (OGD/R) and C57BL/6 male mouse of transient middle artery occlusion followed by 24 h reperfusion (MCAO/R) were established the model of CIR injury. Our results show that BCP remarkably protected against cell death and apoptosis induced by OGD/R, and decreased neurologic injury, infarct volume, and the injury of neurons in CA1 region on MCAO/R mice. In addition, BCP accelerated mitophagy by regulating expression of mitochondrial autophagy marker molecules and the mt-Atp6/Rpl13 ratio (reflecting the relative number of mitochondria), and promoting autophagosome formation compared with OGD/R and MCAO/R groups both in vitro and in vivo. Furthermore, this study revealed that BCP pre-treatment could activate the Pink1/Parkin2 signaling pathway, also with mitophagy activation. To explore the mechanisms, mitochondrial division inhibitor-1 (Mdivi-1) was used to investigate the role of BCP in CIR injury. We found that Mdivi-1 not only decreased BCP-induced facilitation of mitophagy, but also significantly weakened BCP-induced protection against OGD/R and MCAO/R models, which was consistent with levels of Pink1/Parkin2 signaling pathway. Taken together, these results suggest that facilitating mitophagy via Pink1/Parkin2 signaling is essential for the neuroprotective effect of BCP against CIR injury.

Increased level of FAM19A5 is associated with cerebral small vessel disease and leads to a better outcome.

Objective: FAM19A5 plays an essential role in the development and acute or chronic inflammation of the central nervous system. The present study aimed to explore the association between FAM19A5 and cerebral small vessel disease (cSVD). Methods: A total of 344 recent small subcortical infarct (RSSI) patients and 265 healthy controls were included in this study. The difference in the FAM19A5 level between the two groups was compared and the correlation between FAM19A5 and cerebral infarction volume was analyzed. Also, the association between FAM19A5 and the total magnetic resonance imaging (MRI) burden with its imaging characteristics was explored. Moreover, the correspondence of FAM19A5 with the outcome was assessed via Δ National Institutes of Health Stroke Scale score (NIHSS) and the percentage of NIHSS improvement. Results: FAM19A5 was highly expressed in the RSSI group (P = 0.023), showing a positive correlation with cerebral infarction volume (P < 0.01). It was positively correlated with total MRI cSVD burden (P < 0.001) and reflected the severity of white matter hyperintensity (WMH) (P < 0.01) and enlarged perivascular space (EPVS) (P < 0.01), but did not show any association with cerebral microbleed (CMB) and lacune. Moreover, FAM19A5 suggested a larger Δ NIHSS (P = 0.021) and NIHSS improvement percentage (P = 0.007). Conclusion: Serum FAM19A5 was increased in RSSI and positively correlated with the infarct volume. It also reflects the total MRI burden of cSVD, of which the imaging characteristics are positively correlated with WMH and EPVS. In addition, higher FAM19A5 levels reflect better outcomes in RSSI patients.

Plasma chromogranin A levels are associated with acute ischemic stroke with anterior circulation large vessel occlusion.

BACKGROUND AND AIMS: To investigate the relationship between chromogranin A (CgA) levels and acute ischemic stroke (AIS), especially anterior circulation large vessel occlusion (LVO). METHODS AND RESULTS: 587 subjects were included in this study, including 205 AIS patients with anterior circulation LVO and 205 nonocclusive patients, as well as 177 healthy controls. On admission, plasma CgA levels were measured and neurological deficits were assessed by the NIH Stroke Scale. Outcomes were assessed by the modified Rankin Scale at 3 months. The predictive properties of CgA were evaluated by receiver operating characteristic (ROC) curve analysis. Binary logistic analysis assessed the association of CgA levels and AIS or anterior circulation LVO. AIS patients had lower CgA levels than health controls (p < 0.001). Anterior circulation LVO patients had lower CgA levels than nonocclusive patients (p < 0.001). The area under the ROC curve of plasma CgA levels in predicting anterior circulation LVO from AIS was 0.744 and the optimal cutoff value was 15.49 ng/mL with a Youden value of 0.332. Logistic analysis showed that CgA ≤15.49 ng/mL remained an independent risk factor for anterior circulation LVO after adjusting for related factors (OR = 6.519, 95% CI: 3.790-11.214, p < 0.001). CgA was an independent protective factor for mild stroke and good prognosis (p = 0.009, p = 0.005); however, the association disappeared after adjusting for occlusion (p = 0.768, p = 0.335). CONCLUSION: CgA levels were lower in AIS patients, especially in anterior circulation LVO patients. Lower CgA levels are potential biomarker for anterior circulation LVO, and they may indicate good prognosis at 3 months in AIS.

Geraniol-Mediated Suppression of Endoplasmic Reticulum Stress Protects against Cerebral Ischemia-Reperfusion Injury via the PERK-ATF4-CHOP Pathway.

Endoplasmic reticulum (ER) stress plays an important role in cerebral ischemia-reperfusion injury (CIRI). Geraniol has antioxidant, antibacterial, and anti-inflammatory activities. Studies have shown that geraniol has a protective effect against CIRI in rats, but the exact mechanism is unclear. Purpose: The aim of this study was to investigate the protective mechanism of geraniol against CIRI. We established a middle cerebral artery occlusion reperfusion model in rats and a PC12 cell oxygen-glucose deprivation/reoxygenation (OGD/R) model to observe the neuroprotective effects of geraniol. Neurological scoring, 2,3,5-triphenyltetrazolium chloride staining, and hematoxylin and eosin staining were used to evaluate the neuroprotective effects of geraniol against CIRI. ER-stress-related and apoptosis-related protein expression was detected via Western blotting and immunofluorescence. Apoptosis was also detected via TUNEL assays and flow cytometry. The fluorescent detection of intracellular calcium was achieved using fluorescent calcium-binding dyes, and transmission electron microscopy was used to assess the neuronal ultrastructure. Geraniol effectively attenuated cerebral infarction and pathological injury after CIRI, had a protective effect against CIRI, significantly reduced the expression of the ER-stress-related proteins P-PERK, ATF4, CHOP, and GRP78 and the pro-apoptotic protein BAX, increased the expression of the anti-apoptotic protein BCL-2, and reduced the occurrence of apoptosis. In the OGD/R model in PC12 cells, the protective effect of geraniol was the same as that in vivo. Our results suggest that geraniol has a protective effect against ischemic stroke by a mechanism possibly related to ER stress via the PERK-ATF4-CHOP pathway.

Lower uric acid level may be associated with hemorrhagic transformation after intravenous thrombolysis.

BACKGROUND: Previous studies have shown that uric acid (UA) is a powerful water-soluble antioxidant and free radical scavenger for humans. However, the relationship between serum uric acid (SUA) and hemorrhagic transformation (HT) is still controversial. To address this challenge, we aimed to explore the association between serum UA and HT in patients with acute ischemic stroke (AIS) after intravenous thrombolysis (IVT). METHODS: A retrospective analysis was conducted in patients with anterior circulation AIS who underwent IVT at Affiliated Hospital of Qingdao University from 2016 to 2021. HT was evaluated by CT or MRI within 7 days after admission. Baseline demographic, clinical, and laboratory data were compared between the HT and non-HT groups, and between different types of HT groups which were documented according to the European Cooperative Acute Stroke Study III Classification (ECASS III). RESULTS: A total of 727 AIS patients were enrolled, including 112 patients who experienced HT (HT group) and 615 patients who did not experience HT (non-HT group). Patients with HT had significantly lower UA levels compared to those without HT (253.65 ± 97.75 vs 315.97 ± 96.42, p < 0.001); however, there was no significant difference for UA levels in different types of HT (p = 0.907). After adjusting confounders, patients in the fourth UA quartile showed a significant decrease in HT compared with those in the first quartile (OR 0.266, 95% CI 0.107-0.661, p = 0.006). The best cutoff value was identified as 218.5 µmol/L after analysis. CONCLUSIONS: These findings suggest that low levels of UA may be associated with HT after IVT.

Comprehensive Analysis of Peripheral Exosomal circRNAs in Large Artery Atherosclerotic Stroke.

Exosomes are crucial vehicles in intercellular communication. Circular RNAs (circRNAs), novel endogenous noncoding RNAs, play diverse roles in ischemic stroke. Recently, the abundance and stability of circRNAs in exosomes have been identified. However, a comprehensive analysis of exosomal circRNAs in large artery atherosclerotic (LAA) stroke has not yet been reported. We performed RNA sequencing (RNA-Seq) to comprehensively identify differentially expressed (DE) exosomal circRNAs in five paired LAA and normal controls. Further, quantitative real-time PCR (qRT-PCR) was used to verify the RNA-Seq results in a cohort of stroke patients (32 versus 32). RNA-Seq identified a total of 462 circRNAs in peripheral exosomes; there were 25 DE circRNAs among them. Additionally, circRNA competing endogenous RNA (ceRNA) network and translatable analysis revealed the potential functions of the exosomal circRNAs in LAA progression. Two ceRNA pathways involving 5 circRNAs, 2 miRNAs, and 3 mRNAs were confirmed by qRT-PCR. In the validation cohort, receiver operating characteristic (ROC) curve analysis identified two circRNAs as possible novel biomarkers, and a logistic model combining two and four circRNAs increased the area under the curve compared with the individual circRNAs. Here, we show for the first time the comprehensive expression of exosomal circRNAs, which displayed the potential diagnostic and biological function in LAA stroke.

Mesenchymal stem-cell-derived exosomal miR-145 inhibits atherosclerosis by targeting JAM-A.

Atherosclerosis is a chronic inflammatory disease associated with the development of plaques that can be converted into an acute clinical event by thrombosis or plaque rupture. Mesenchymal stem cells (MSCs) exhibit therapeutic effects for the treatment of various diseases, including atherosclerosis. In this study, we show that microRNA-145 (miR-145) is associated with atherosclerosis by microRNA sequencing and bioinformatics analysis. MSC-derived miR-145-rich exosomes could efficiently deliver miR-145 from MSCs to human umbilical vein endothelial cells (HUVECs). Treatment of miR-145-rich exosomes could downregulate JAM-A, inhibit migration in vitro, and reduce atherosclerotic plaque in vivo. Our study suggests that MSC-derived miR-145-rich exosomes have great potential for atherosclerosis prevention.

K63 ubiquitin chains target NLRP3 inflammasome for autophagic degradation in ox-LDL-stimulated THP-1 macrophages.

Inflammation, especially involving the NLRP3 inflammasome, is critical to atherosclerotic plaque formation. Enhanced autophagy can inhibit the development of atherosclerosis, and recent studies have revealed that NLRP3 inflammasome can be degraded by autophagy in atherosclerosis. In the present study, we established a foam-cell model to investigate the impact of oxidized low density lipoproteins (ox-LDLs) on autophagy and the inflammasome in atherosclerosis-related inflammation. We observed that ox-LDLs activated NLRP3 inflammasomes in macrophages and restricted autophagy in a time-and dose-dependent manner. We further observed through immunoprecipitation and siRNA knockdown that autophagic degradation of the NLRP3 inflammasome is dependent on K63 polyubiquitation of its NLRP3 subunit and subsequent binding by the adaptor protein p62. Our findings uncover a mechanism by which autophagy inhibits inflammation in atherosclerosis and the role of K63 in that process.

Fucoidan Inhibits NLRP3 Inflammasome Activation by Enhancing p62/SQSTM1-Dependent Selective Autophagy to Alleviate Atherosclerosis.

NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation contributes to the progression of atherosclerosis, and autophagy inhibits inflammasome activation by targeting macrophages. We investigated whether fucoidan, a marine sulfated polysaccharide derived from brown seaweeds, could reduce NLRP3 inflammasome activation by enhancing sequestosome 1 (p62/SQSTM1)-dependent selective autophagy to alleviate atherosclerosis in high-fat-fed ApoE-/- mice with partial carotid ligation and differentiated THP-1 cells incubated with oxidized low-density lipoprotein (oxLDL). Fucoidan significantly ameliorated lipid accumulation, attenuated progression of carotid atherosclerotic plaques, deregulated the expression of NLRP3 inflammasome, autophagy receptor p62, and upregulated microtubule-associated protein light chain 3 (LC3)-II/I levels. Transmission electron microscopy and GFP-RFP-LC3 lentivirus transfection further demonstrated that fucoidan could activate autophagy. Mechanistically, fucoidan remarkably inhibited NLRP3 inflammasome activation, which was mostly dependent on autophagy. The inhibitory effects of fucoidan on NLRP3 inflammasome were enhanced by autophagy activator rapamycin (Rapa) and alleviated by autophagy inhibitor 3-methyladenine (3-MA). Fucoidan promoted the colocalization of NLRP3 and p62. Knockdown of p62 and ATG5 by small interfering RNA significantly reduced the inhibitory effects of fucoidan treatment on NLRP3 inflammasome. The data suggest that fucoidan can inhibit NLRP3 inflammasome activation by enhancing p62/SQSTM1-dependent selective autophagy to alleviate atherosclerosis.

MicroRNA-155 promotes the ox-LDL-induced activation of NLRP3 inflammasomes via the ERK1/2 pathway in THP-1 macrophages and aggravates atherosclerosis in ApoE-/- mice.

BACKGROUND: Nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome activation can induce the secretion of IL-1ß and IL-18 and after promoting the development of atherosclerosis. MiR-155 is an important microRNA that modulates inflammation in atherosclerosis, but the role of miR-155 in the regulation of the NLRP3 inflammasome is still unknown. METHODS: The atherosclerosis model was set up using ApoE-/- mice, and the lentiviral vector (LV) was used to interfere the expression of miR-155. HE stains was used for plaque morphology, immunohistochemistry (IHC) and western blot were used for protein expression quantification. We used oxidized low-density lipoprotein (ox-LDL) to incubate PMA-preprocessed THP-1 macrophages and detected NLRP3 inflammasome activation and ERK1/2 phosphorylation by western blot and Enzyme-linked immunosorbent assay. RESULTS: HE stains showed that the intravascular plaques in the miR-155-up group were remarkably increased, compared with negative control (NC) group. Results of IHC showed that the expression of caspase-1 and IL-1ß in the miR-155-up group was the highest of four groups, consist with the Western blot analysis. The results of in vitro experiment show that ox-LDL promoted NLRP3 inflammasome activation and ERK1/2 phosphorylation. Blocking the ERK1/2 pathway could inhibit ox-LDL-induced NLRP3 inflammasome activation. Moreover, we found that the overexpression of miR-155 promoted the activation of the ox-LDL-induced NLRP3 inflammasome, which could also be blocked by the ERK inhibitor U0126. CONCLUSIONS: MiR-155 aggravates the carotid AS lesion in ApoE-/- mice and exerts a regulatory effect on NLRP3 inflammasome activation in ox-LDL-induced macrophages via the ERK1/2 pathway.

MicroRNA-181a regulates the activation of the NLRP3 inflammatory pathway by targeting MEK1 in THP-1 macrophages stimulated by ox-LDL.

Atherosclerosis (AS) is a chronic inflammatory disease that is characterized by the deposition of lipids in the vascular wall and the formation of foam cells. Macrophages play a critical role in the development of this chronic inflammation. An increasing amount of research shows that microRNAs affect many steps of inflammation. The goal of our study was to investigate the regulatory effect of miR-181a on the NLRP3 inflammasome pathway and explore its possible mechanism. Compared with the control group, the expression of miR-181a was downregulated in the carotid tissue of AS group mice, while the expression of MEK1 and NLRP3-related proteins was upregulated significantly. In vitro, when THP-1 macrophages were stimulated with oxidized low-density lipoprotein (ox-LDL), the expression of miR-181a was decreased, the MEK/ERK/NF-κB inflammatory pathways were activated and the expression of NLRP3 inflammasome-related proteins was upregulated. Exogenous overexpression of miR-181a downregulated the activation of the MEK/ERK/NF-κB pathway and decreased the expression of NLRP3 inflammasome-related proteins (such as NLRP3, caspase-1, interleukin-18 [IL-18], IL-1ß, etc). Exogenous miR-181a knockdown showed the opposite results to those of overexpression group. A luciferase reporter assay proved that miR-181a inhibited the expression of MEK1 by binding to its 3'-untranslated region. When we knocked down miR-181a and then treated cells with U0126 before ox-LDL stimulation, we found that U0126 reversed the increased activation of the MEK/ERK/NF-κB pathway and upregulation of NLRP3 inflammasome-related proteins (NLRP3, caspase-1, IL-18, IL-1ß) that resulted from miR-181a knockdown. Our study suggests that miR-181a regulates the activation of the NLRP3 inflammatory pathway by altering the activity of the MEK/ERK/NF-κB pathway via targeting of MEK1.

LPS induces CXCL16 expression in HUVECs through the miR-146a-mediated TLR4 pathway.

Endothelial inflammation characterizes the early stages of atherosclerosis. CXCL16 is a protein that functions as both a chemokine and adhesion molecule, playing a crucial role in the pathogenesis of atherosclerosis. However, it is uncertain if LPS, a major inducer of inflammation, affects CXCL16 expression in endothelial cells and whether miR-146a, a negative regulator of atherosclerosis, participates in this process. The present study showed that exposure of human umbilical vein endothelial cells (HUVECs) to LPS induced the overexpression of CXCL16, TLR4 and NF-κB, and this induction was blocked by the TLR4 inhibitor TAK-242. In addition, LPS induced the upregulation of miR-146a in HUVECs. Overexpression or inhibition of miR-146a either inhibited or increased the LPS-induced expression CXCL16, TLR4 and NF-κB protein production, respectively. Additionally, miR-146a-induced CXCL16 expression was blocked by TAK-242. Thus, in this study, we demonstrate that LPS stimulates CXCL16 expression via the TLR4/NF-κB signaling pathway, and simultaneously, miR-146 negatively regulates LPS-induced CXCL16 expression through a TLR4-dependent mechanism.

LncRNA MALAT1 promotes oxidized low-density lipoprotein-induced autophagy in HUVECs by inhibiting the PI3K/AKT pathway.

Emerging evidence suggests that long noncoding RNAs (lncRNAs) are involved in many biological processes, such as cell growth, differentiation, apoptosis, and autophagy. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), highly expressed in endothelial cells, is well conserved and implicated in endothelial cell migration and proliferation. However, whether MALAT1 participates in oxidized low-density lipoprotein (ox-LDL)-induced autophagy regulation in human umbilical vein endothelial cells (HUVECs) remains unknown. In this study, we observed that autophagy was upregulated and MALAT1 expression was markedly increased in HUVECs treated with ox-LDL. The ox-LDL-induced autophagy of HUVECs is significantly associated with the PI3K/AKT pathway. Furthermore, we found that MALAT1 overexpression inhibited PI3K, Akt and p70S6K phosphorylation and downregulated RHEB expression, simultaneously increasing ox-LDL-induced autophagy. MALAT1 silencing caused higher phosphorylated PI3K, Akt and p70S6K levels, upregulated RHEB expression and markedly suppressed autophagy. These results indicated that lncRNA MALAT1 promotes ox-LDL-induced autophagy in HUVECs partly through the PI3K/AKT signaling pathway.

Associations of miR-146a, miR-149, miR-196a2, and miR-499 Polymorphisms with Ischemic Stroke in the Northern Chinese Han Population.

BACKGROUND Recently, miR-146a C>G, miR- 149 T>C, miR-196a2 T>C and miR-499 A>G polymorphisms have been associated with susceptibility to many diseases, including ischemic stroke (IS). However, results have been reported inconsistency in IS, especially in the Chinese population. This study aimed to investigate the polymorphisms of the 4 miRNAs and IS risk in the Chinese population. MATERIAL AND METHODS We used a case-control study to explore these associations in 396 patients with IS and 378 healthy controls. According to TOAST standards, the selected patients were divided into subgroups: the large artery atherosclerosis (LAA) subgroup and the small artery occlusion (SAO) subgroup. The method of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was used to detect the genotypes. RESULTS The miR-146a C>G polymorphism was remarkably different (CC vs. CG+GG: P=0.027; CC+CG vs. GG: P=0.020; C vs. G: P=0.006). The miR-149 T>C polymorphism was also remarkably different (TT vs. TC+CC: P=0.017; TT+TC vs. CC: P=0.020; T vs. C: P=0.004). The miR-146a and miR-149 polymorphisms were also remarkably different in the LAA subgroup (P<0.05). However, we did not find an association of miR-196a2 T>C or miR-499 A>G polymorphisms with IS (P>0.05); we did not find any association in the LAA subgroup or the SAO subgroup (P>0.05). CONCLUSIONS Our study suggested that miR-146a C>G and miR-149 T>C polymorphisms might remarkably increase the risk of IS, which might be mainly associated with an increased risk in LAA stroke; however, the miR-196a2 T>C and miR-499 A>G polymorphisms might not be associated with IS risk in the northern Chinese Han population.

MicroRNA­155 promotes ox­LDL­induced autophagy in human umbilical vein endothelial cells by targeting the PI3K/Akt/mTOR pathway.

Endothelial cell autophagy has a protective role in inhibiting inflammation and preventing the development of atherosclerosis, which may be regulated by microRNA (miR)­155. The present study aimed to investigate the mechanisms of autophagy in the development of atherosclerosis. Human umbilical vein endothelial cells model in vitro and using oxidized low­density lipoprotein (ox­LDL) stimulated cells to simulate the atherosclerosis. MiR­155 mimics, miR­155 inhibitors, and a negative control were respectively transfected in human umbilical vein endothelial cells to analyzed alterations in the expression of miR­155. It was demonstrated that overexpression of miR­155 promoted autophagic activity in oxidized low­density lipoprotein­stimulated human umbilical vein endothelial cells, whereas inhibition of the expression of miR­155 reduced autophagic activity. Overexpression of miR­155 revealed that it regulated autophagy via the phosphatidylinositol­3 kinase (PI3K)/RAC­α serine/threonine­protein kinase (Akt)/mechanistic target of rapamycin pathway (mTOR) signaling pathway. A luciferase reporter assay demonstrated that miR­155 directly bound to the PI3K catalytic subunit a and Ras homolog enriched in brain 3'­untranslated region and inhibited its luciferase activity. Therefore, the results of the present study suggested that miR­155 promoted autophagy in vascular endothelial cells and that this may have occurred via targeting of the PI3K/Akt/mTOR pathway. Thus, miR­155 may be considered as a potential therapeutic target for the treatment of atherosclerosis.

Associations of CXCL16, miR­146a and miR­146b in atherosclerotic apolipoprotein E­knockout mice.

Atherosclerosis is the primary cause of cardiovascular and cerebrovascular diseases. Recent studies have revealed that C­X­C motif chemokine ligand 16 (CXCL16), microRNA (miR)­146a and miR­146b may have important roles in atherosclerotic diseases. However, the associations of CXCL16, miR­146a and miR­146b in atherosclerotic diseases in vivo remain unclear. Previous studies have demonstrated that miR­146a and miR­146b may negatively regulate the toll like receptor (TLR4)/nuclear factor (NF)­κB signaling pathway to repress the inflammatory response. The present study investigated the associations of CXCL16, miR­146a and miR­146b in atherosclerotic apolipoprotein E (ApoE)­/­ mice in vivo. The expression levels of CXCL16, TLR4/NF­κB signaling pathway, miR­146a and miR­146b in the control and atherosclerotic ApoE­/­ mice were investigated via reverse transcription­quantitative polymerase chain reaction and western blot analysis. The present study demonstrated that the expression of CXCL16 was significantly upregulated in atherosclerotic ApoE­/­ mice compared with control ApoE­/­ mice. The expression levels of TRL4, interleukin­1 receptor­associated kinase 1, tumor necrosis factor receptor associated factor 6, NF­κB, tumor necrosis factor­α and interleukin­1ß were also significantly upregulated in atherosclerotic ApoE­/­ mice compared with control mice. However, the present study revealed that the expression levels of miR­146a and miR­146b were significantly downregulated in atherosclerotic ApoE­/­ mice compared with control ApoE­/­ mice. Overall, the results of the present study suggested that CXCL16 may regulate the TRL4/NF­κB/CXCL16 signaling pathway, and that miR­146a and miR­146b may negatively regulate CXCL16 via this pathway in atherosclerosis in vivo.