Alisol A 24-Acetate, a Triterpenoid Derived from Alisma orientale, Inhibits Ox-LDL-Induced Phenotypic Transformation and Migration of Rat Vascular Smooth Muscle Cells through Suppressing ERK1/2 Signaling.

Alisol A 24-acetate, a triterpenoid extracted from Alisma orientale, has shown antiatherosclerotic actions. The purpose of this study was to evaluate the inhibition of alisol A 24-acetate on oxidized low-density lipoprotein (Ox-LDL)-induced phenotypic transformation and migration of rat vascular smooth muscle cells (VSMCs), and to explore the underlying mechanisms. VSMCs were pretreated with alisol A 24-acetate and a specific extracellular signal-regulated kinase (ERK) inhibitor, U0126, and then stimulated with 50 mg/l Ox-LDL in vitro. The expression of VSMC phenotypic marker SM22α was determined using immunocytochemistry, and the migration of VSMCs was detected using a scratch-wound healing assay. The expression of matrix metalloproteinase (MMP)-9, MMP-2, phosphorylated ERK1/2 (pERK1/2) and total ERK was determined. Ox-LDL treatment caused a reduction in SM22α expression, VSMC transformation to the synthetic phenotype, increased MMP-2 and MMP-9 synthesis, the extension of VSMC migration distance and the upregulation of pERK1/2 expression, while the addition of alisol A 24-acetate or U0126 resulted in the elevation of SM22α expression, VSMC transformation to the contractile phenotype, a reduction in MMP-2 and MMP-9 expression, the shortening of cell migration distance and decreased pERK1/2 expression. The results of this study demonstrate that alisol A 24-acetate effectively reverses the phenotypic transformation and inhibits the migration of VSMCs, which may be associated with the suppression of the ERK1/2 signaling pathway.

The Compatibility of Alisma and Atractylodes Affects the Biological Behaviours of VSMCs by Inhibiting the miR-128-5p/p21 Gene.

Objective: The compatibility of Alisma and Atractylodes (AA) has been estimated to exhibit antiatherosclerotic effects, but the mechanism remains unclear. This study aimed to identify the role of AA in oxidized low-density lipoprotein (ox-LDL)-induced vascular smooth muscle cell (VSMC) behaviours and to explore the effects of microRNAs (miRNAs). Methods: A scratch wound-healing assay was used to detect the migration of VSMCs, and immunocytochemistry and western blotting for SM22ɑ were used to evaluate phenotypic transformation. Bromodeoxyuridine (BrdU) immunocytochemistry and flow cytometry were applied to detect the proliferation of VSMCs. miRNA microarray profiling was performed using Lianchuan biological small RNA sequencing analysis. VSMCs were transfected with the miR-128-5p mimic and inhibitor, and the migration, phenotypic modulation, and proliferation of VSMCs were investigated. The 3'UTR-binding sequence site of miR-128-5p on the p21 gene was predicted and assessed by luciferase assays. Result: AA and the extracellular regulated protein kinase 1/2 (ERK1/2) blocker U0126 markedly inhibited migration, elevated smooth muscle 22α (SM22α) expression, repressed VSMC proliferation, elevated miR-466f-3p and miR-425-3p expression, and suppressed miR-27a-5p and miR-128-5p expression in ox-LDL-induced VSMCs. miR-128-5p targets the tissue inhibitor of metalloproteinases (TIMPs), silent information regulator 2 (SIRT2), peroxisome proliferator-activated receptor (PPAR), and p21 genes, which are linked to the behaviours of VSMCs. The miR-128-5p mimic promoted the migration and proliferation of VSMCs and suppressed p21, p27, and SM22ɑ expression. The inhibitor increased p21, p27, and SM22ɑ expression and repressed the migration, phenotypic transformation, and proliferation of VSMCs. miR-128-5p directly targeted the 3'UTR-binding sequences of the p21 gene, negatively regulated p21 expression, and supported the proliferation of VSMCs. Conclusion: Our research showed that the migration, phenotypic transformation, and proliferation of ox-LDL-induced VSMCs were repressed by AA through inhibiting miR-128-5p by targeting the p21 gene, which may provide an effective option for the treatment of atherosclerosis.

Emerging blood exosome-based biomarkers for preclinical and clinical Alzheimer's disease: a meta-analysis and systematic review.

Blood exosomes, which are extracellular vesicles secreted by living cells into the circulating blood, are regarded as a relatively noninvasive novel tool for monitoring brain physiology and disease states. An increasing number of blood cargo-loaded exosomes are emerging as potential biomarkers for preclinical and clinical Alzheimer's disease. Therefore, we conducted a meta-analysis and systematic review of molecular biomarkers derived from blood exosomes to comprehensively analyze their diagnostic performance in preclinical Alzheimer's disease, mild cognitive impairment, and Alzheimer's disease. We performed a literature search in PubMed, Web of Science, Embase, and Cochrane Library from their inception to August 15, 2020. The research subjects mainly included Alzheimer's disease, mild cognitive impairment, and preclinical Alzheimer's disease. We identified 34 observational studies, of which 15 were included in the quantitative analysis (Newcastle-Ottawa Scale score 5.87 points) and 19 were used in the qualitative analysis. The meta-analysis results showed that core biomarkers including Aß1-42, P-T181-tau, P-S396-tau, and T-tau were increased in blood neuron-derived exosomes of preclinical Alzheimer's disease, mild cognitive impairment, and Alzheimer's disease patients. Molecules related to additional risk factors that are involved in neuroinflammation (C1q), metabolism disorder (P-S312-IRS-1), neurotrophic deficiency (HGF), vascular injury (VEGF-D), and autophagy-lysosomal system dysfunction (cathepsin D) were also increased. At the gene level, the differential expression of transcription-related factors (REST) and microRNAs (miR-132) also affects RNA splicing, transport, and translation. These pathological changes contribute to neural loss and synaptic dysfunction. The data confirm that the above-mentioned core molecules and additional risk-related factors in blood exosomes can serve as candidate biomarkers for preclinical and clinical Alzheimer's disease. These findings support further development of exosome biomarkers for a clinical blood test for Alzheimer's disease. This meta-analysis was registered at the International Prospective Register of Systematic Reviews (Registration No. CRD4200173498, 28/04/2020).

Association between Ngb polymorphisms and ischemic stroke in the Southern Chinese Han population.

BACKGROUND: Neuroglobin (Ngb), one of novel members of the globin superfamily, is expressed predominantly in brain neurons, and appears to modulate hypoxic-ischemic insults. The mechanisms underlying Ngb-mediated neuronal protection are still unclear. For it is one of the candidate protective factors for ischemic stroke, we conducted a case-control study to clarify the association of Ngb polymorphisms with ischemic stroke in the Southern Chinese Han population. METHODS: 355 cases and 158 controls were recruited. With brain imaging, cases were subdivided into large-artery atherosclerosis (LVD) and small-vessel occlusion (SVD) stroke. PCR amplified all the four exons of Ngb and flanking intron sequence for each exon. Genotyping for Ngb was achieved by direct sequencing and mismatched PCR-RFLP. Polymorphisms were studied both individually and as haplotypes in each group and subgroup which subdivided according to gender or age. RESULTS: Two intronic polymorphisms 89+104 c>t and 322-110 (6a)>5a were identified. The allele frequency of 89+104 t was decreased in stroke cases. The protective effect seems to be more pronounced in subgroups of female patients and age > 60 years. Also, we have confirmed decreased LDL-C level and reduced hypertension and hypercholesterolemia in 89+104 t allele carriers. In contrast, the 322-110 (6a)>5a genotype distribution was similar between cases and controls. However, the haplotype 89+104 c>t/322-110 (6a)>5a was related with LVD and SVD stroke. The haplotype c-5a was more frequent in both LVD and SVD groups while t-6a was more frequent in controls. CONCLUSION: Ngb polymorphism 89+104 t had protective effects on LVD and SVD in the Southern Chinese Han population. A "hitchhiking" effect was observed for the 89+104 t/322-110 (6a) genotype combination especially for LVD.

Novel mutation in the ABCA1 gene identified in a chinese patient with dementia and atherothrombotic cerebral infarction.

BACKGROUND: To date, 81 mutations of ATP-binding cassette transporter 1 (ABCA1) have been reported. However, no ABCA1 mutation has been reported in the Chinese population. METHODS: We used direct sequencing to screen for ABCA1 mutations in 72 patients with both atherosclerotic cerebral infarction (ACI) and plasma high-density lipoprotein cholesterol (HDL-C) < 0.8 mmol/l. The functionality of the mutation was verified using 200 unrelated controls and 76 patients with ACI and normal HDL-C by PCR-RFLP analysis. RESULTS: One patient with dementia prior to ACI was found to carry the heterozygous Y2206D mutation, which has not been reported previously. The patient had a medical history of atherosclerosis in the coronary and carotid arteries going back 40 years and splenohepatomegalia for 13 years, with a low plasma HDL-C level (0.66 mmol/l) and apolipoprotein A1 level (0.61 mmol/l). During the past decade, he had developed symptoms of dementia. Sixteen months prior to the study, he was admitted to hospital for an ACI. CONCLUSION: The results suggest that this patient is most likely a patient with familial hypoalphalipoproteinemia and that the Y2206D mutation may be associated with not only a lower level of HDL-C, but also with dementia.

Inhibition of ERK1/2 improves lipid balance in rat macrophages via ABCA1/G1 and CD36.

ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1), and macrophage scavenger receptor, cluster of differentiation (CD)36, function as key mediators of cholesterol efflux and influx from macrophages. In addition, they are associated with foam cell formation and the development of atherosclerosis (AS). The aim of the present study was to investigate the effects of extracellular signal-regulated kinases 1/2 (ERK1/2) inhibition on lipid balance in oxidized-low-density lipoprotein (Ox-LDL)-stimulated rat macrophages, and to examine the role of ERK1/2 inhibitors in AS. Rat peritoneal macrophages were treated with Ox-LDL alone or in combination with an ERK1/2 inhibitor, U0126, and untreated cells served as controls. Ox-LDL-induced lipid accumulation was detected by DiI fluorescence and oil red O staining. In addition, the mRNA and protein expression levels of ABCA1, ABCG1 and CD36 were determined using polymerase chain reaction and western blotting, respectively. Treatment with Ox-LDL significantly increased lipid accumulation and upregulated the mRNA and protein expression levels of ABCA1, ABCG1 and CD36 in macrophages. The addition of U0126 resulted in a marked reduction of lipid deposition, upregulation of ABCA1/G1 expression and suppression of CD36 expression in Ox-LDL-stimulated macrophages. The results of the present study indicated a novel association between ERK1/2 signaling and lipid metabolism, thus suggesting that inhibition of ERK1/2 may be considered a promising therapeutic strategy against AS.

TAT-mediated delivery of neuroglobin attenuates apoptosis induced by oxygen-glucose deprivation via the Jak2/Stat3 pathway in vitro.

OBJECTIVES: Neuroglobin (Ngb), an identified globin in vertebrate brain, is a potential novel protective protein against brain ischemia. In our previous study, the human immunodeficiency virus trans-activator of transcription (TAT) protein transduction domain successfully delivered exogenous Ngb into neurons in the mouse, and protected the brain from cerebral ischemia-induced apoptosis. The aim of this study is to investigate the role of TAT-Ngb in attenuating oxygen-glucose deprivation (OGD) induced apoptosis and to explore the possible mechanism. METHODS: Nerve growth factor (NGF)-induced PC12 cells were divided into (1) the control group, (2) the OGD group (just OGD), (3) the Ngb treatment group (OGD and Ngb treatment), and (4) the TAT-Ngb treatment group (OGD and TAT-Ngb treatment). Cell viability and apoptosis were assessed by the MTT assay and the AnnexinV/propidium iodide (PI) staining, respectively. The mitochondrial transmembrane potential was measured by JC-1 staining. Caspase-3, Bcl-2, Bax, Stat3, Jak2, and Akt were determined by western blot analysis. RESULTS: Trans-activator of transcription effectively delivered Ngb into NGF-induced PC12 cells. Neuroglobin-mediated neuroprotection rescued cultured cells from OGD. We also confirmed previous findings that TAT-Ngb inhibited mitochondrial apoptosis following OGD. Inhibition of apoptosis by Ac-DEVD-CHO showed that caspase-3 was a crucial factor in OGD-induced apoptosis cascades. AG490, a specific Jak2 inhibitor, attenuated the protective effects of TAT-Ngb. The TAT-Ngb promoted expression of the anti-apoptotic protein Bcl-2 through the Jak2/Stat3 signal pathway, and inhibited apoptosis by blocking caspase-3 activation, while the Jak-Akt-Stat3 signal network was not involved. CONCLUSION: Our results demonstrate that TAT-Ngb can protect neuron-like cells against OGD-induced apoptosis by activating the Jak2/Stat3 pathway.

TAT-mediated delivery of neuroglobin protects against focal cerebral ischemia in mice.

Neuroglobin (Ngb), a newly identified globin in vertebrate brain, has been suggested to be able to protect against brain hypoxic-ischemic injury. However, owing to its large size, the impermeability of the blood-brain barrier (BBB) to Ngb limits its application in brain injury. Recently, the 11-amino-acid human immunodeficiency virus trans-activator of transcription (TAT) protein transduction domain was shown to successfully deliver macromolecules into the brain. This study explored whether the TAT-Ngb fusion protein can cross the BBB and protect the brain from cerebral ischemia. The TAT-Ngb fusion protein generated from Escherichia coli BL21 (DE3) was efficiently delivered into mice brain tissues by intravenous injection as demonstrated by immunohistochemistry and Western blotting. Two groups of mice were treated with filamentous middle cerebral artery occlusion (MCAO) for 30min or 2h followed by reperfusion. Each group was then divided into sub-groups and was injected intravenously with TAT-Ngb, Ngb, or saline respectively before MCAO or immediately after reperfusion. Compared with the Ngb- and saline-treated group, the group with TAT-Ngb treated 2h before MCAO showed significantly less brain infarct volume and had better neurologic outcomes (p<0.05). Furthermore, a TAT-Ngb injection following a 30-min MCAO treatment significantly increased neuronal survival in the striatum (p<0.05). Our results demonstrated that the exogenous Ngb fusion protein containing the TAT protein transduction domain could be efficiently transduced into neurons in the mouse and protect the brain from mild or moderate ischemic injury.

The R219K polymorphism in the ATP-binding cassette transporter 1 gene has a protective effect on atherothrombotic cerebral infarction in Chinese Han ethnic population.

The association of R219K and V825I polymorphisms of ABCA1 gene with cerebral infarction has been rarely reported. Here we wish to address this issue. A total of 476 subjects from Chinese Han ethnic population were investigated, including 152 control individuals and 324 patients with cerebral infarction. Genotyping of R219K and V825I were performed by PCR-RFLP analysis. Data were analyzed using a statistical package. The R219K genotype frequency distributions were significantly different between patients with atherothrombotic cerebral infarction (ACI) and control individuals, with fewer KK genotypes and more RR genotypes in ACI patients (chi(2)=9.89, P<0.01). The K allele is less frequent among ACI patients than in controls (chi(2)=9.16, P<0.005). A significant association of KK with decreased ACI risk was exhibited, especially in male patients, aged patients and individuals with hypertension. These results indicate that the K allele of R219K polymorphism is an independent protective factor against ACI. In addition, though there is no association of V825I with ACI, this polymorphism may have certain synergistic effect with hypertension in susceptibility to ACI.

Electroacupuncture improves neurological deficits and enhances proliferation and differentiation of endogenous nerve stem cells in rats with focal cerebral ischemia.

OBJECTIVES: This study was carried out to observe the effect of electroacupuncture (EA) on neurological deficits, proliferation and differentiation of nerve stem cells (NSCs) in adult rats with middle cerebral artery occlusion (MCAO) and to study its possible role in the treatment of cerebral ischemic injury. METHODS: A rat model of MCAO was established and interfered with EA. On days 4, 7, 14 and 21 after ischemic injury, neurological deficits were scored. On days 4, 7, 14 and 21 after injury, effect of EA interference on the proliferation and differentiation of rat NSCs was observed with BrdU/NeuN and BrdU/GFAP immunofluorescence double labeling. RESULTS: A significant difference was found in the scores of rat neurological deficits between the EA and model groups 7, 14 and 21 days after cerebral ischemic injury (p<0.05). BrdU positive cells were found in the subventricular zone (SVZ) 4, 7, 14 and 21 days after ischemic injury. The number of positive BrdU cells in the SVZ reached its peak 7 days after injury and was greater in the EA group than in the model group 7 and 14 days after injury (p<0.05). The number of BrdU/GFAP doubly labeled positive cells in the SVZ was greater in the EA group than in the model group 7 and 14 days after ischemic injury (p=0.012 and p=0.025, respectively). There was no difference in the number of BrdU/NeuN doubly labeled positive cells 4, 7 and 14 days in the striatum, but a significant difference 21 days (p=0.033) after ischemic injury between the two groups. DISCUSSION: Cerebral ischemic injury induces proliferation of NSCs, some of which will differentiate into both astroglia and neurons. EA may promote cells proliferation, stimulate the proliferating cells to differentiate into astroglia and mature into neurons, which may be one of the important reasons why EA can alleviate neurological deficits.