Transcriptome-wide N6-methyladenosine methylation profile of atherosclerosis in mice.

BACKGROUND: Atherosclerosis (AS) is a critical pathological event during the progression of cardiovascular diseases. It exhibits fibrofatty lesions on the arterial wall and lacks effective treatment. N6-methyladenosine (m6A) is the most common modification of eukaryotic RNA and plays an important role in regulating the development and progression of cardiovascular diseases. However, the role of m6A modification in AS remains largely unknown. Therefore, in this study, we explored the transcriptome distribution of m6A modification in AS and its potential mechanism. METHODS: Methylation Quantification Kit was used to detect the global m6A levels in the aorta of AS mice. Western blot was used to analyze the protein level of methyltransferases. Methylated RNA immunoprecipitation with next-generation sequencing (MeRIP-seq) and RNA sequencing (RNA-seq) were used to obtain the first transcriptome range analysis of the m6A methylene map in the aorta of AS mice, followed by bioinformatics analysis. qRT-PCR and MeRIP-qRT-PCR were used to measure the mRNA and m6A levels in target genes. RESULTS: The global m6A and protein levels of methyltransferase METTL3 were significantly increased in the aorta of AS mice. However, the protein level of demethylase ALKBH5 was significantly decreased. Through MeRIP-seq, we obtained m6A methylation maps in AS and control mice. In total, 26,918 m6A peaks associated with 13,744 genes were detected in AS group, whereas 26,157 m6A peaks associated with 13,283 genes were detected in the control group. Peaks mainly appeared in the coding sequence (CDS) regions close to the stop codon with the RRACH motif. Moreover, functional enrichment analysis demonstrated that m6A-containing genes were significantly enriched in AS-relevant pathways. Interestingly, a negative correlation between m6A methylation abundance and gene expression level was found through the integrated analysis of MeRIP-seq and RNA-seq data. Among the m6A-modified genes, a hypo-methylated but up-regulated (hypo-up) gene Fabp5 may be a potential biomarker of AS. CONCLUSIONS: Our study provides transcriptome-wide m6A methylation for the first time to determine the association between m6A modification and AS progression. Our study lays a foundation for further exploring the pathogenesis of AS and provides a new direction for the treatment of AS.

Apolipoprotein E mimetic peptide COG1410 alleviates blood­brain barrier injury in a rat model of ischemic stroke.

Blood­brain barrier (BBB) damage is one of the main causes of poor outcomes and increased mortality rates following cerebral ischemia­reperfusion injury. Apolipoprotein E (ApoE) and its mimetic peptide have been previously reported to exhibit potent neuroprotective properties in various central nervous system disease models. Therefore, the present study aimed to investigate the possible role of the ApoE mimetic peptide COG1410 in cerebral ischemia­reperfusion injury and its potential underlying mechanism. Male SD rats were subjected to 2 h middle cerebral artery occlusion followed by 22 h reperfusion. Evans blue leakage and IgG extravasation assays results revealed that COG1410 treatment significantly reduced BBB permeability. In addition, in situ zymography and western blotting were used to prove that COG1410 was able to downregulate the activities of MMPs and upregulate the expression of occludin in the ischemic brain tissue samples. Subsequently, COG1410 was found to significantly reverse microglia activation while also suppressing inflammatory cytokine production, according to immunofluorescence signal of Iba­1 and CD68 and protein expression of COX­2. Consequently, this neuroprotective mechanism mediated by COG1410 was further tested using the BV2 cell line in vitro, which was exposed to oxygen glucose deprivation followed by reoxygenation. The mechanism of COG1410 was found to be mediated, as least partly, through the activation of triggering receptor expressed on myeloid cells 2. In conclusion, the data suggest that COG1410 can alleviate BBB injury and neuroinflammation following ischemic stroke.

LINC01123 promotes cell proliferation and migration via regulating miR-1277-5p/KLF5 axis in ox-LDL-induced vascular smooth muscle cells.

The pathophysiological mechanism of carotid atherosclerosis (CAS) involves endothelial cell dysfunction, vascular smooth muscle cells (VSMCs), and macrophage activation, which ultimately leads to fibrosis of the vessel wall. lncRNA works weightily in the formation of CAS, but the function and mechanism of lncRNA LINC01123 in stable plaque formation are still equivocal. We collected blood samples from 35 CAS patients as well as 33 healthy volunteers. VSMCs treated with oxidized low-density lipoprotein (ox-LDL) were utilized as the CAS cell models. We applied qRT-PCR for detecting LINC01123, miR-1277-5p and KLF5 mRNA expression, CCK-8 method and BrdU test for determining cell proliferation, Transwell test for measuring cell migration, as well as Western blot for assaying KLF5 protein expression. Dual-luciferase reporter experiment was adopted for assessing the interaction between LINC01123 and miR-1277-5p, as well as KLF5 and miR-1277-5p. LINC01123 and KLF5 expression were dramatically up-regulated, while miR-1277-5p expression was down-regulated in CAS patients and ox-LDL-induced CAS cell models. Overexpressed LINC01123 notedly promoted VSMCs migration and proliferation. LINC01123 knockdown repressed cell proliferation and migration. Also, LINC01123 targeted miR-1277-5p and down-regulated its expression, while miR-1277-5p could negatively regulate KLF5 expression. LINC01123 is highly expressed in CAS patients, and promotes cell proliferation and migration via regulating miR-1277-5p/KLF5 axis in ox-LDL-induced VSMCs. It might be involved in the fibrous plaque formation.

Spinosin and 6'''­Feruloylspinosin protect the heart against acute myocardial ischemia and reperfusion in rats.

Investigating active compounds from Chinese herbal medicine that can rescue myocardial cells is a good approach to preserve cardiac function. Several herbal formulae that containing Semen Ziziphi Spinosae (SZS), also called Suanzaoren in Chinese, are clinically effective in the treatment of patients with acute myocardial infarction (AMI). The present study aimed to investigate the cardioprotective effects of spinosin and 6'''­feruloylspinosin, two flavonoid glycosides from SZS, in a rat model of myocardial ischemia and reperfusion. The left anterior descending artery (LAD) was occluded to induce myocardial ischemia. Spinosin or 6'''­feruloylspinosin (5 mg/kg) was intraperitoneally injected into rats 30 min before LAD ligation. The protein levels of myocardial enzymes in the serum, the extent of tissue injury and the rate of apoptosis were examined after AMI in rats with or without pretreatment with spinosin or 6'''­feruloylspinosin. Western blotting was performed to investigate the potential mechanisms underlying the function of these two flavonoid glycosides. The present results suggested that pretreatment with spinosin or 6'''­feruloylspinosin significantly attenuated myocardial tissue injury, and reduced myocardial enzyme release and cell apoptosis in AMI rats. In addition, spinosin treatment increased light chain 3B­II and 6'''­feruloylspinosin, and reduced p62, indicating that autophagy was promoted after drug treatments. Treatments of spinosin and 6'''­feruloylspinosin led to the reduction of glycogen synthase kinase­3ß (GSK3ß) phosphorylation at Tyr216, and the increase of peroxisome proliferator­activated receptor γ coactivator (PGC)­1α and its downstream signaling proteins, including nuclear factor (erythroid­derived 2)­like 2 (Nrf2) and hemeoxygenase1 (HO­1). The present data suggested that SZS flavonoids could protect myocardial cells against acute heart ischemia­reperfusion, probably via the inhibition of GSK3ß, which increased autophagy and the activity of the PGC­1α/Nrf2/HO­1 pathway.

Outbreak of Vaccinia Virus Infection from Occupational Exposure, China, 2017.

Human infections with vaccinia virus (VACV), mostly from laboratory accidents or contact with infected animals, have occurred since smallpox was eradicated in 1980. No recent cases have been reported in China. We report on an outbreak of VACV from occupational exposure to rabbit skins inoculated with VACV.

Geniposide regulates the miR-101/MKP-1/p38 pathway and alleviates atherosclerosis inflammatory injury in ApoE-/- mice.

Atherosclerosis (AS) is the common pathological basis of chronic cardiovascular diseases and is associated with inflammation and lipid metabolism dysfunction. Geniposide, the main active ingredient of Gardenia jasminoides Ellis fruit, exhibits a variety of anti-inflammatory and anti-oxidative functions; however, its role in AS remains unclear. The aim of this study was to investigate the mechanisms of geniposide in alleviating inflammation and thereby attenuating the development of AS. ApoE-/- mice were fed a high fat diet to induce AS and were treated with geniposide (50 mg/kg) for 12 weeks. Blood glucose and lipid levels were measured by biochemical analysis. H&E, Masson and Oil red O staining were performed to observe morphological changes and lipid deposition in the aorta and liver. Serum inflammatory cytokines were detected by ELISA. Dual-luciferase reporter gene assay was used to verify the target relationship between microRNA-101 (miR-101) and mitogen-activated protein kinase phosphatase-1 (MKP-1). The levels of miR-101, p-p38, and MKP-1 in the aorta were detected by qPCR and western blotting. The anti-inflammatory effect of geniposide in vitro was investigated in the RAW264.7 macrophage cell line. A miR-101 mimic and an inhibitor were used to study the effect of miR-101 on regulating the expression of the target MKP-1 and the downstream inflammatory cytokines. Geniposide treatment reduced lipid levels and plaque size in the mouse model of AS. Geniposide downregulated miR-101 to upregulate MKP-1 and suppress the production of inflammatory factors in vitro and in vivo. Geniposide suppressed the levels of inflammatory factors in the presence of the miR-101 mimic, whereas no obvious effect was observed in the miR-101 inhibitor group. We concluded that geniposide reduced the plaque size and alleviated inflammatory injury in ApoE-/- mice and RAW264.7 cells. The specific anti-inflammatory mechanism was related to the miR-101/ MKP-1/p38 signaling pathway.

IRAK-M Deficiency Exacerbates Ischemic Neurovascular Injuries in Experimental Stroke Mice.

Background: Innate immune response to neuronal death is one of the key events of the pathogenesis of ischemic brain injury. Interleukin-1 receptor-associated kinase (IRAK)-M, encoded by gene Irak3, negatively regulates toll-like receptor signaling by interacting with the MyD88-IRAK-4-IRAK-1 complex and blocking the phosphorylation and dissociation of IRAK-1. Its function in the ischemic stroke is unknown. Objective: This study aims to investigate whether IRAK-M deficiency could exacerbate neuroinflammation and neurovascular injuries during cerebral ischemia and reperfusion. Methods: Male C57BL/6 mice and Irak3 knockout mice were subjected to 45 min of middle cerebral artery occlusion and 4 or 24 h of reperfusion. Transcription of Irak3 gene was evaluated by quantitative real-time PCR (qRT-PCR). Then, infarct volume, neurological score, brain water content, and Evans blue leakage were compared between knock-out and wild-type mice after reperfusion. Through the observation of gross brain specimen after cerebral ischemia, the incidence of hemorrhage transformation was compared between KO and WT mice. To explore underlying signaling pathways involved in IRAK-M deficiency, major proinflammatory cytokines and NF-κB signaling were measured by qRT-PCR and Western blot. Results: The expression of IRAK-M peaked at 1 h after reperfusion, and then gradually decreased within the first 24 h, which was abolished by blocking the expression of hypoxia induced factor 1α. IRAK-M deficiency increased infarct volume, brain edema, the incidence of hemorrhage transformation, and the permeability of blood-brain barrier. In addition, the NF-κB-mediated expressions of proinflammatory cytokines and the activation of microglia in the ipsilateral brain from knock-out mice were much higher than those in wild-type littermates. Conclusion: IRAK-M deletion exacerbates neurovascular damages which are related to the pronounced activation of NF-κB signaling and neuroinflammatory responses during cerebral ischemia-reperfusion in mice. Our study indicates that IRAK-M has neuroprotective effect and has potential to facilitate the development of new pharmaceuticals that reduce neurovascular complications.

[Effect of Electroacupuncture on the Expression of Hippocampal eNSCs in MCAO Model Rats].

Objective To observe the effects of electroacupuncture (EA) on hippocampal en- dogenous neural stem cells (eNSCs) expression of middle cerebral artery occlusion (MCAO) model rats after cerebral ischemia-reperfusion (I/R) at different time points, and to observe possible mechanisms of EA for keeping away from damage in acute cerebral infarction (ACI). Methods MCAO model was pre- pared in male SPF grade SD rats by suture method. Totally 90 rats were divided into the sham-operated group, the model group, and the EA group according to random number table, 30 in each group. Rats in the sham-operated group only received surgical trauma. Rats in the model group only received MCAO I/R injury. Rats in the EA group received EA at Baihui (DU20) and Dazhui (DU14) , once per day, 30 min each time. Nerve defects of rats were tested by neural function defect scale at day 1 , 7, 14 of treatment, respectively. Meanwhile, 6 rats were executed randomly from each group. Their hippocampus tissues were isolated. Then the proliferation and differentiation expression of eNSCs in the hippocampus area were detected by immunofluorescence method. Results (1) The scores of nerve function defect scale: The scores of the model group increased at day 1, 7, 14 of treatment, being higher as compared with those of the sham-operated group (P <0. 05). The scores of the EA group were lower than those of the model group at day 1, 7, 14 of treatment (P <0. 05). (2) The expression of BrdU positive cells: Com- pared with the sham-operated group, the expression of BrdU positive cells in the model group were in- creased at day 1, 7, 14 of treatment (P <0. 05). Compared with the model group at each time points, the expression of BrdU positive cells in the EA group were increased more at day 1, 7, 14 of treatment (P < 0. 05). (3) The expression of Nestin positive cells: The expression of Nestin positive cells were in- creased more in the model group than in the sham-operated group at day 1 , 7, 14 of treatment (P < 0. 05). Compared with the model group, the expression of Nestin positive cells increased more in the EA group, but only with statistical difference at day 7 of treatment (P <0. 05). (4) the expression of DCX positive cells: the expression of DCX positive cells were increased more in the model group than in the sham-operated group at day 1 and 7 of treatment (P <0. 05). Compared with the model group, the ex- pression of DCX positive cells were increased more in the EA group at day 7 and 14 of treatment (P < 0. 05). (5) the expression of NeuN positive cells: The expression NeuN of positive cells were increased more in the model group than in the sham-operated group at day 1, 7, and 14 of treatment, but only with statistical difference at day 14 of treatment (P <0. 05). Compared with the model group, the expression of NeuN positive cells were increased more obviously, but only with statistical difference at day 1 and 14 of treatment (P <0.05). (6) the expression of GFAP positive cells: The expression of GFAP positive cells increased more obviously in the model group than in the sham-operated group at day 1 , 7, and 14 of treatment (P <0. 05). Compared with the model group, the expression of GFAP positive cells were not obviously increased in the EA group, but only with statistical difference at day 14 of treatment (P <0. 05). Conclusions The proliferation and differentiation of eNSCs exist in the hippocampus area after cerebral I/R in MCAO model rats. EA could improve the recovery of damaged nerve function. Its possible mecha- nism might lie in that EA could promote the proliferation and differentiation of eNSCs in hippocampus area, inhibit excessive differentiation of eNSCs into astrocytes , promote differentiation of eNSCs into neu- rons, and improve regeneration of nerve cells.

Electroacupuncture attenuates cervical spinal cord injury following cerebral ischemia/reperfusion in stroke-prone renovascular hypertensive rats.

Cerebral ischemia induces injury, not only in the ischemic core and surrounding penumbra tissues, but also in remote areas such as the cervical spinal cord. The aim of the present study was to determine the effects of electroacupuncture (EA) on cervical spinal cord injury following cerebral ischemia/reperfusion in stroke-prone renovascular hypertensive (RHRSP) rats. The results demonstrated that neuronal loss, which was assayed by Nissl staining in the cervical spinal cords of RHRSP rats subjected to transient middle cerebral artery occlusion (MCAO), was markedly decreased by EA stimulation at the GV20 (Baihui) and GV14 (Dazhui) acupoints compared with that in rats undergoing sham stimulation. Quantitative polymerase chain reaction and western blot analysis demonstrated that EA stimulation blocked the MCAO-induced elevated protein expression levels of glial fibrillary acidic protein and amyloid precursor protein in the cervical spinal cord at days 24 and 48. To further investigate the mechanism underlying the neuroprotective role of EA stimulation, the protein expression levels of Nogo-A and Nogo-66 receptor-1 (NgR1), two key regulatory molecules for neurite growth, were recorded in each group. The results revealed that EA stimulation reduced the MCAO-induced elevation of Nogo-A and NgR1 protein levels at day 14 and 28 in RHRSP rats. Therefore, the results demonstrated that EA reduced cervical spinal cord injury following cerebral ischemia in RHRSP rats, indicating that EA has the potential to be developed as a therapeutic treatment agent for cervical spinal cord injury following stroke.