Metformin protects high glucose­cultured cardiomyocytes from oxidative stress by promoting NDUFA13 expression and mitochondrial biogenesis via the AMPK signaling pathway.

Tissue damage in diabetes is at least partly due to elevated reactive oxygen species production by the mitochondrial respiratory chain during hyperglycemia. Sustained hyperglycemia results in mitochondrial dysfunction and the abnormal expression of mitochondrial genes, such as NADH: Ubiquinone oxidoreductase subunit A13 (NDUFA13). Metformin, an AMP­activated protein kinase (AMPK) activator, protects cardiomyocytes from oxidative stress by improving mitochondrial function; however, the exact underlying mechanisms are not completely understood. The aim of the present study was to investigated the molecular changes and related regulatory mechanisms in the response of H9C2 cardiomyocytes to metformin under high glucose conditions. H9C2 cells were subjected to CCK­8 assay to assess cell viability. Reactive oxygen species generation was measured with DCFH­DA assay. Western blotting was used to analyze the expression levels of NDUFA13, AMPK, p­AMPK and GAPDH. Reverse transcription­quantitative PCR was used to evaluate the expression levels of mitochondrial genes and transcription factors. It was observed that metformin protected H9C2 cardiomyocytes by suppressing high glucose (HG)­induced elevated oxidative stress. In addition, metformin stimulated mitochondrial biogenesis, as indicated by increased expression levels of mitochondrial genes (NDUFA1, NDUFA2, NDUFA13 and manganese superoxide dismutase) and mitochondrial biogenesis­related transcription factors [peroxisome proliferator­activated receptor­gamma coactivator­1α, nuclear respiratory factor (NRF)­1, and NRF­2] in the metformin + HG group compared with the HG group. Moreover, metformin promoted mitochondrial NDUFA13 protein expression via the AMPK signaling pathway, which was abolished by pretreatment with the AMPK inhibitor, Compound C. The results suggested that metformin protected cardiomyocytes against HG­induced oxidative stress via a mechanism involving AMPK, NDUFA13 and mitochondrial biogenesis.

MicroRNA-153 impairs hippocampal synaptic vesicle trafficking via downregulation of synapsin I in rats following chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion (CCH) promotes the development of Alzheimer's pathology. However, whether and how CCH impairs the synaptic vesicle trafficking is still unclear. In the present study, we found that the hippocampal glutamatergic vesicle trafficking was impaired as indicated by a significant shortened delayed response enhancement (DRE) phase in CA3-CA1 circuit and decreased synapsin I in CCH rats suffering from bilateral common carotid artery occlusion (2VO). Further study showed an upregulated miR-153 in the hippocampus of 2VO rats. In vitro, overexpression of miR-153 downregulated synapsin I by binding the 3'UTRs of SYN1 mRNAs, which was prevented by its antisense AMO-153 and miRNA-masking antisense oligodeoxynucleotides (SYN1-ODN). In vivo, the upregulation of miR-153 elicited similar reduced DRE phase and synapsin I deficiency as CCH. Furthermore, miR-153 knockdown rescued the downregulated synapsin I and shortened DRE phase in 2VO rats. Our results demonstrate that CCH impairs hippocampal glutamatergic vesicle trafficking by upregulating miR-153, which suppresses the expression of synapsin I at the post-transcriptional level. These results will provide important references for drug research and treatment of vascular dementia.

MicroRNA-153 impairs presynaptic plasticity by blocking vesicle release following chronic brain hypoperfusion.

BACKGROUND: Chronic brain hypoperfusion (CBH) is closely related to Alzheimer's disease (AD) and vascular dementia (VaD). Meanwhile, synaptic pathology plays a prominent role in the initial stage of AD and VaD. However, whether and how CBH impairs presynaptic plasticity is currently unclear. METHODS: In the present study, we performed a battery of techniques, including primary neuronal culture, patch clamp, stereotaxic injection of the lentiviral vectors, morris water maze (MWM), dual luciferase reporter assay, FM1-43 fluorescence dye evaluation, qRT-PCR and western blot, to investigate the regulatory effect of miR-153 on hippocampal synaptic vesicle release both in vivo and in vitro. The CBH rat model was generated by bilateral common carotid artery ligation (2VO). RESULTS: Compared to sham rats, 2VO rats presented decreased field excitatory postsynaptic potential (fEPSP) amplitude and increased paired-pulse ratios (PPRs) in the CA3-CA1 pathway, as well as significantly decreased expression of multiple vesicle fusion-related proteins, including SNAP-25, VAMP-2, syntaxin-1A and synaptotagmin-1, in the hippocampi. The levels of microRNA-153 (miR-153) were upregulated in the hippocampi of rats following 2VO surgery, and in the plasma of dementia patients. The expression of the vesicle fusion-related proteins affected by 2VO was inhibited by miR-153, elevated by miR-153 inhibition, and unchanged by binding-site mutation or miR masks. FM1-43 fluorescence images showed that miR-153 blunted vesicle exocytosis, but this effect was prevented by either 2'-O-methyl antisense oligoribonucleotides to miR-153 (AMO-153) and miR-masking of the miR-153 binding site in the 3' untranslated region (3'UTR) of the Snap25, Vamp2, Stx1a and Syt1 genes. Overexpression of miR-153 by lentiviral vector-mediated miR-153 mimics (lenti-pre-miR-153) decreased the fEPSP amplitude and elevated the PPR in the rat hippocampus, whereas overexpression of the antisense molecule (lenti-AMO-153) reversed these changes triggered by 2VO. Furthermore, lenti-AMO-153 attenuated the cognitive decline of 2VO rats. CONCLUSIONS: Overexpression of miR-153 controls CBH-induced presynaptic vesicle release impairment by posttranscriptionally regulating the expression of four vesicle release-related proteins by targeting the 3'UTRs of the Stx1a, Snap25, Vamp2 and Syt1 genes. These findings identify a novel mechanism of presynaptic plasticity impairment during CBH, which may be a new drug target for prevention or treatment of AD and VaD. Video Abstract.

Impaired Left Atrial Performance Resulting From Age-Related Arial Fibrillation Is Associated With Increased Fibrosis Burden: Insights From a Clinical Study Combining With an in vivo Experiment.

Background: Atrial fibrillation (AF) is increasingly considered an age-related degenerative disease, whose process is associated with the development of impaired left atrial (LA) performance. However, the subtle dynamic changes of LA performance in AF during aging have yet to be fully elucidated. Atrial fibrosis is a key substrate for the development of AF, but the progression of fibrosis during aging and its relationship with LA dysfunction need to be further explored. Methods: A total of 132 control individuals and 117 persistent AF patients were prospectively studied. Subjects were further stratified into three age groups (age group 1: younger than 65 years, age group 2: between 65 and 79 years old, and age group 3: older than 80 years). The two-dimensional speckle tracking imaging was carried out for analyzing the alterations in LA function underlying LA remodeling, whereas electroanatomic mapping was performed to investigate LA fibrosis burden. In animal study, aged mice and young mice served as research subjects. Echocardiography and histological staining were used to assess LA performance and fibrosis burden, respectively. Results: Echocardiography showed progressive increases in LA dimension and LA stiffness index, and progressive decreases in LA global longitudinal strain and LA strain rates with advancing age in both AF and control cohorts, which was more prominent in AF cohort. Electroanatomic mapping showed progressive decrease in mean LA voltage and progressive increases in LA surface area, low-voltage area %, and LA volume with advancing age, whereas more significant alterations were observed in AF patients. Moreover, left atrial global longitudinal strain was positively correlated with mean LA voltage, whereas LA stiffness index was negatively related to mean LA voltage. In animal experiment, increased LA size and pulmonary artery dimension as well as longer P-wave duration and more prominent LA fibrosis were found in aged mice. Conclusions: This study provides new evidence of subtle changes in structure and performance of left atrium and their association with atrial fibrosis in both AF and non-AF subjects during physiological aging. In addition, our study also provides normal values for LA structure and performance in both AF and non-AF conditions during aging. These measurements may provide an early marker for onset of AF and LA adverse remodeling.

The chemical constituents from the active fractions of Eleutherine bulbosa with their antimicrobial activity.

Six compounds including three new polyketide ones named eleubosas A-C (1-3) were isolated from the active frations of Eleutherine bulbosa. Their structures were elucidated by extensive spectroscopic methods, including NMR, MS and IR spectroscopic analyses data. All the isolates were evaluated against three pathogenic bacteria, Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa, and the results showed that compounds 1 and 2 displayed moderate inhibitory activities against E. coli with MIC values both 12.5 µg/mL, which are consistent with the clinical applications and need further studies.

Ursane-type triterpenoids from the roots of Rosa multiflora with their anti-inflammatory activity.

Nine ursane-type triterpenoids including three new ones 2α, 19α-dihydroxyurs-3-O-acetyltormentic acid (1), 1α, 2α, 3α, 20ß-tetrahydroxyurs -13(18)-en-28-oic acid (2), and 2α, 3α, 20ß, 24-tetrahydroxyurs-13(18)-en-28-oic acid (3) were isolated from the roots of Rosa multiflora. Their structures were elucidated by extensive spectroscopic methods, including NMR, MS, and IR spectroscopic analyses data. All the isolates were evaluated for their anti-inflammatory activity in vitro and the results showed that compounds 1-9 displayed moderate inhibitory activity with IC50 values ranging from 24.7 to 86.2 µM compared with the postitive control Amino guanidine (IC50 4.3 µM).[Formula: see text].

Metoprolol prevents neuronal dendrite remodeling in a canine model of chronic obstructive sleep apnea.

Obstructive sleep apnea (OSA) is closely associated with central nervous system diseases and could lead to autonomic nerve dysfunction, which is often seen in neurodegenerative diseases. Previous studies have shown that metoprolol prevents several chronic OSA-induced cardiovascular diseases through inhibiting autonomic nerve hyperactivity. It remains unclear whether chronic OSA can lead to dendritic remodeling in the brain, and whether metoprolol affects the dendritic remodeling. In this study we investigated the effect of metoprolol on dendrite morphology in a canine model of chronic OSA, which was established in beagles through clamping and reopening the endotracheal tube for 4 h every other day for 12 weeks. OSA beagles were administered metoprolol (5 mg· kg-1· d-1). The dendritic number, length, crossings and spine density of neurons in hippocampi and prefrontal cortices were assessed by Golgi staining. And the protein levels of hypoxia-inducible factor-1α (HIF-1α) and brain-derived neurotrophic factor (BDNF) were measured by Western blotting. We showed that chronic OSA successfully induced significant brain hypoxia evidenced by increased HIF-1α levels in CA1 region and dentate gyrus of hippocampi, as well as in prefrontal cortex. Furthermore, OSA led to markedly decreased dendrite number, length and intersections, spine loss as well as reduced BDNF levels. Administration of metoprolol effectively prevented the dendritic remodeling and spine loss induced by chronic OSA. In addition, administration of metoprolol reversed the decreased BDNF, which might be associated with the metoprolol-induced neuronal protection. In conclusion, metoprolol protects against neuronal dendritic remodeling in hippocampi and prefrontal cortices induced by chronic OSA in canine.

Overexpression of miR-1 in the heart attenuates hippocampal synaptic vesicle exocytosis by the posttranscriptional regulation of SNAP-25 through the transportation of exosomes.

BACKGROUND: The link between cardiac diseases and cognitive deterioration has been accepted from the concept of "cardiogenic dementia", which was proposed in the late 1970s. However, the molecular mechanism is unclarified. METHODS: The two animal models used in this study were cardiac-specific overexpression of microRNA-1-2 transgenic (Tg) mice and a myocardial infarction mouse model generated by left coronary artery ligation (LCA). First, we observed the microRNA-1 (miR-1) level and synaptic vesicles (SV) distribution in the hippocampus using in situ hybridization and transmission electron microscopy (TEM) and evaluated the expression of vesicle exocytosis related proteins by western blotting. Second, we used dual luciferase reporter assay as well as antagonist and miRNA-masking techniques to identify the posttranscriptional regulatory effect of miR-1 on the Snap25 gene. Third, FM1-43 staining was performed to investigate the effect of miR-1 on synaptic vesicle exocytosis. Lastly, we used GW4869 to inhibit the biogenesis and secretion of exosomes to determine the transportation effect of exosomes for miR-1 from the heart to the brain. RESULTS: Compared with the levels in age-matched WT mice, miR-1 levels were increased in both the hearts and hippocampi of Tg mice, accompanied by the redistribution of SVs and the reduction in SV exocytosis-related protein SNAP-25 expression. In vitro studies showed that SNAP-25 protein expression was down- or upregulated by miR-1 overexpression or inhibition, respectively, however, unchanged by miRNA-masking the 3'UTR of the Snap25 gene. SV exocytosis was inhibited by miR-1 overexpression, which could be prevented by co-transfection with an anti-miR-1 oligonucleotide fragment (AMO-1). The knockdown of miR-1 by hippocampal stereotaxic injection of AMO-1 carried by a lentivirus vector (lenti-pre-AMO-1) led to the upregulation of SNAP-25 expression and prevented SV concentration in the synapses in the hippocampi of Tg mice. The application of GW4869 significantly reversed the increased miR-1 level in the blood and hippocampi as well as reduced the SNAP-25 protein levels in the hippocampi of both Tg and LCA mice. CONCLUSION: The overexpression of miR-1 in the heart attenuated SV exocytosis in the hippocampus by posttranscriptionally regulating SNAP-25 through the transportation of exosomes. This study contributes to the understanding of the relationship between cardiovascular disease and brain dysfunction.

Knockdown of MicroRNA-1 in the Hippocampus Ameliorates Myocardial Infarction Induced Impairment of Long-Term Potentiation.

BACKGROUNDS/AIMS: It has been reported that myocardial infarction (MI) is a risk factor for vascular dementia. However, the molecular mechanism remains largely unknown. METHODS: MI mice were generated by ligation of the left coronary artery (LCA) for 4 weeks. Passive and active avoidance tests were performed to evaluate the cognitive ability of MI mice. A theta-burst stimulation (TBS) protocol was applied to elicit long-term potentiation (LTP) of the perforant pathway-dentate gyrus synapse (PP-DG). Western blot analysis was employed to assess protein levels. RESULTS: In this study, we demonstrated that after 4 weeks of MI, C57BL/6 mice had significantly impaired memory. Compared with the sham group, in vivo physiological recording in the MI group revealed significantly decreased amplitude of population spikes (PS) with no effect on the latency and duration of the stimulus-response curve. The amplitude of LTP was markedly decreased in the MI group compared with the sham group. Further examination showed that the expression of the TBS-LTP-related proteins BDNF, GluA1 and phosphorylated GluA1 were all decreased in the MI group compared with those in the sham group. Strikingly, all these changes were prevented by hippocampal stereotaxic injection of an anti-miR-1 oligonucleotide fragment carried by a lentivirus vector (lenti-pre-AMO-1). CONCLUSION: MI induced cognitive decline and TBS-LTP impairment, and decreased BDNF and GluA1 phosphorylation levels from overexpression of miR-1ated were involved in this process.

[Study on lignans from Xanthii Fructus].

This project is to investigate lignans from the dried fruits of Xanthium sibiricum (Xanthii Fructus). The chemical constituents were extract by 70% ethanol and isolated by silica gel, ODS, Sephadex LH-20, MCI column chromatography. Based on comparison of their spectral data with those reported in literature, they were elucidated as (-)-pinoresinol (1), balanophonin A (2), diospyrosin (3), dehydrodiconiferyl alcohol (4), 2-(4-hydroxy-3-methoxyphenyl)-3-(2-hydroxy-5-methoxyphenyl)-3-oxo-1-propanol (5), (-)-simulanol (6), (-)-7R,8S-dehydrodiconiferyl alcohol (7), chushizisin E (8), dihydrodehydrodiconiferyl alcohol (9), 7R,8S-dihydrodehydrodiconiferyl alcohol 4-O-ß-D-glucopyranoside (10), erythro-1,2-bis(4-hydroxy-3-methoxyphenyl)-1,3-propanediol (11), leptolepisol D (12), 8-O-4' neolignan 4-O-ß-glucopyranoside (13), (-)-1-O-ß-D-glucopyranosyl-2-{2-methoxy-4-[1-(E)-propen-3-ol]phenoxyl}-propane-3-ol(14), 1-(4-hydroxy-3-methoxy)-phenyl-2-[4-(1,2,3-trihydroxypropyl)-2-methoxy]-phenoxy-1,3-propandiol (15), threo-dihydroxy dehydrodiconiferyl alcohol (16), (-)-(2R)-1-O-ß-D-glucopyranosyl-2-{2-methoxy-4-[(E)-formylviny1]phenoxyl} propane-3-ol (17). Compound 2-17 were isolated from the genus Xanthium for the first time. Compound 1 were isolated form Xanthii Fructus for the first time.

Cardiac-Specific Overexpression of Silent Information Regulator 1 Protects Against Heart and Kidney Deterioration in Cardiorenal Syndrome via Inhibition of Endoplasmic Reticulum Stress.

BACKGROUND/AIMS: Increased endoplasmic reticulum (ER) stress contributes to development of cardiorenal syndrome (CRS), and Silent Information Regulator 1 (SIRT1), a class III histone deacetylase, may have protective effects on heart and renal disease, by reducing ER stress. We aimed to determine if SIRT1 alleviates CRS through ER stress reduction. METHODS: Wild type mice (n=37), mice with cardiac-specific SIRT1 knockout (n=29), or overexpression (n=29), and corresponding controls, were randomized into four groups: sham MI (myocardial infarction) +sham STNx (subtotal nephrectomy); MI+sham STNx; sham MI+STNx; and MI+STNx. To establish the CRS model, subtotal nephrectomy (5/6 nephrectomy, SNTx) and myocardial infarction (MI) (induced by ligation of the left anterior descending (LAD) coronary artery) were performed successively to establish CRS model. At week 8, the mice were sacrificed after sequential echocardiographic and hemodynamic studies, and then pathology and Western-blot analysis were performed. RESULTS: Neither MI nor STNx alone significantly influenced the other healthy organ. However, in MI groups, STNx led to more severe cardiac structural and functional deterioration, with increased remodeling, increased BNP levels, and decreased EF, Max +dp/dt, and Max -dp/dt values than in sham MI +STNx groups. Conversely, in STNx groups, MI led to renal structural and functional deterioration, with more severe morphologic changes, augmented desmin and decreased nephrin expression, and increased BUN, SCr and UCAR levels. In MI+STNx groups, SIRT1 knockout led to more severe cardiac structural and functional deterioration, with higher Masson-staining score and BNP levels, and lower EF, FS, Max +dp/dt, and Max -dp/dt values; while SIRT1 overexpression had the opposite attenuating effects. In kidney, SIRT1 knockout resulted in greater structural and functional deterioration, as evidenced by more severe morphologic changes, higher levels of UACR, BUN and SCr, and increased desmin and TGF-ß expression, while SIRT1 overexpression resulted in less severe morphologic changes and increased nephrin expression without significant influence on BUN or SCr levels. The SIRT1 knockout but not overexpression resulted in increased myocardial expression of CHOP and GRP78. Cardiac-specific SIRT1 knockout or overexpression resulted in increased or decreased renal expression of CHOP, Bax, and p53 respectively. CONCLUSIONS: Myocardial SIRT1 activation appears protective to both heart and kidney in CRS models, probably through modulation of ER stress.

A Rodent Model for Chronic Brain Hypoperfusion Related Diseases: Permanent Bilateral Occlusion of the Common Carotid Arteries (2VO) in Rats.

Permanent occlusion of bilateral common carotid arteries (2VO) in rat is considered as a suitable animal model to mimic chronic brain hypoperfusion status, which is proved to be a risk factor to precede the Alzheimer's disease or/and vascular dementia. In this protocol, we describe how to successfully ligate the bilateral common carotid arteries covered by anterior cervical muscle group, and provide the details for understanding the surgical procedures of 2VO.

MicroRNA-195 prevents dendritic degeneration and neuron death in rats following chronic brain hypoperfusion.

Impaired synaptic plasticity and neuron loss are hallmarks of Alzheimer's disease and vascular dementia. Here, we found that chronic brain hypoperfusion (CBH) by bilateral common carotid artery occlusion (2VO) decreased the total length, numbers and crossings of dendrites and caused neuron death in rat hippocampi and cortices. It also led to increase in N-terminal ß-amyloid precursor protein (N-APP) and death receptor-6 (DR6) protein levels and in the activation of caspase-3 and caspase-6. Further study showed that DR6 protein was downregulated by miR-195 overexpression, upregulated by miR-195 inhibition, and unchanged by binding-site mutation and miR-masks. Knockdown of endogenous miR-195 by lentiviral vector-mediated overexpression of its antisense molecule (lenti-pre-AMO-miR-195) decreased the total length, numbers and crossings of dendrites and neuron death, upregulated N-APP and DR6 levels, and elevated cleaved caspase-3 and caspase-6 levels. Overexpression of miR-195 using lenti-pre-miR-195 prevented these changes triggered by 2VO. We conclude that miR-195 is involved in CBH-induced dendritic degeneration and neuron death through activation of the N-APP/DR6/caspase pathway.

MicroRNA-27a Promotes Inefficient Lysosomal Clearance in the Hippocampi of Rats Following Chronic Brain Hypoperfusion.

Chronic brain hypoperfusion (CBH) induces the accumulation of abnormal cellular proteins, accompanied by cognitive decline, and the autophagic-lysosomal system is abnormal in dementia. Whether CBH accounts for autophagic-lysosomal neuropathology remains unknown. Here, we show that CBH significantly increased the number of autophagic vacuoles (AVs) with high LC3-II levels, but decreased SQSTM1 and cathepsin D levels in the hippocampi of rats following bilateral common carotid artery occlusion (2VO) for 2 weeks. Further studies showed that microRNA-27a (Mir27a) was upregulated at 2 weeks compared with the sham group. Additionally, LAMP-2 proteins were downregulated by Mir27a overexpression, upregulated by Mir27a inhibition, and unchanged by binding-site mutations or miR-masks, indicating that lamp-2 is the target of Mir27a. Knockdown of endogenous Mir27a prevented the reduction of LAMP-2 protein expression as well as the accumulation of AVs in the hippocampi of 2VO rats. Overexpression of Mir27a induced, while the knockdown of Mir27a reduced, the accumulation of AVs and the LC3-II level in cultured neonatal rat neurons. The results revealed that CBH in rats at 2 weeks could induce inefficient lysosomal clearance, which is regulated by the Mir27a-mediated downregulation of LAMP-2 protein expression. These findings provide an insight into a novel molecular mechanism of autophagy at the miRNA level.

New phenylpropanoid derivatives from the fruits of Xanthium sibiricum and their anti-inflammatory activity.

The fruits of Xanthium sibiricum Patr yielded five phenylpropanoid derivatives, named as xanthiumnolics A-E (1-5). Their structures were elucidated by spectroscopic analysis and comparison with literature data. The isolated ones were tested for their anti-inflammatory activities on lipopolysaccharide (LPS)-induced nitric oxide (NO) production in RAW 264.7, and compound 5 showed strong inhibitory activities with IC50 value of 8.73µM.

A new phytoecdysteroid from the roots of Achyranthes bidentata Bl.

A new phytoecdysteroid compound, named Niuxixinsterone D (1), together with two known phytoecdysteroid compounds (2 and 3) were isolated from Achyranthes bidentata Bl.. The structure of the new compound was elucidated by extensive spectral analysis, including HR-ESI-MS, 1D and 2D NMR methods. Compounds 1-3 were tested for their inhibitory effects against LPS-induced NO production in RAW 264.7 macrophages, and compound 1 and 3 exhibited anti-neuroinflammatory activity with inhibited 29.7 and 26.0% NO production.

Knockdown of microRNA-195 contributes to protein phosphatase-2A inactivation in rats with chronic brain hypoperfusion.

Reduction of protein phosphatase-2A (PP2A) activity is a common clinical feature of Alzheimer's disease and vascular dementia. In this study, we observed that chronic brain hypoperfusion induced by bilateral common carotid artery occlusion of rats led to PP2A inactivation based on the increase in tyrosine-307 phosphorylation and leucine-309 demethylation of PP2AC and the depression in PP2ABα. Knockdown of miR-195 using overexpression of its antisense molecule oligonucleotide (pre-AMO-miR-195) delivered by a lentivirus (lenti-pre-AMO-miR-195) increased tyrosine-307 phosphorylation and decreased both PP2ABα expression and leucine-309 methylation; these effects were prevented by the overexpression of miR-195 using lenti-pre-miR-195 and controlled by an increase in methylesterase (PME-1) and a decrease in leucine carboxyl methyltransferase-1. In vitro studies demonstrated that miR-195 regulated PME-1 expression by binding to the Ppme1 gene 3'-untranslated region (3'UTR) domain. Masking the miR-195 binding sites in the amyloid precursor protein (APP) and ß-site APP cleaving enzyme 1 genes prevented miR-195-induced leucine carboxyl methyltransferase-1 elevation. We concluded that the miR-195 downregulation in chronic brain hypoperfusion involved PP2A inactivity, which was mediated by the post-transcriptional regulation PME-1, APP, and ß-site APP cleaving enzyme 1 expression.

MicroRNA-9 induces defective trafficking of Nav1.1 and Nav1.2 by targeting Navß2 protein coding region in rat with chronic brain hypoperfusion.

BACKGROUND: Previous studies have demonstrated that the trafficking defects of Nav1.1/Nav1.2 are involved in the dementia pathophysiology. However, the detailed mechanisms are not fully understood. Moreover, whether the impaired miRNAs regulation linked to dementia is a key player in sodium channel trafficking disturbance remains unclear. The cognitive impairment induced by chronic cerebral ischemia through chronic brain hypoperfusion (CBH) is likely reason to precede dementia. Therefore, our goal in the present study was to examine the role of microRNA-9 (miR-9) in regulating Nav1.1/Nav1.2 trafficking under CBH generated by bilateral common carotid artery occlusion (2VO). RESULTS: The impairment of Nav1.1/Nav1.2 trafficking and decreased expression of Navß2 were found in the hippocampi and cortices of rats following CBH generated by bilateral 2VO. MiR-9 was increased in both the hippocampi and cortices of rats following CBH by qRT-PCR. Intriguingly, miR-9 suppressed, while AMO-miR-9 enhanced, the trafficking of Nav1.1/Nav1.2 from cytoplasm to cell membrane. Further study showed that overexpression of miR-9 inhibited the Navß2 expression by targeting on its coding sequence (CDS) domain by dual luciferase assay. However, binding-site mutation or miR-masks failed to influence Navß2 expression as well as Nav1.1/Nav1.2 trafficking process, indicating that Navß2 is a potential target for miR-9. Lentivirus-mediated miR-9 overexpression also inhibited Navß2 expression and elicited translocation deficits to cell membrane of Nav1.1/Nav1.2 in rats, whereas injection of lentivirus-mediated miR-9 knockdown could reverse the impaired trafficking of Nav1.1/Nav1.2 triggered by 2VO. CONCLUSIONS: We conclude that miR-9 may play a key role in regulating the process of Nav1.1/Nav1.2 trafficking via targeting on Navß2 protein in 2VO rats at post-transcriptional level, and inhibition of miR-9 may be a potentially valuable approach to prevent Nav1.1/Nav1.2 trafficking disturbance induced by CBH.

Activation of Cdk5/p25 and tau phosphorylation following chronic brain hypoperfusion in rats involves microRNA-195 down-regulation.

Chronic brain hypoperfusion (CBH) is a common clinical feature of Alzheimer's disease and vascular dementia, but the underlying molecular mechanism is unclear. Our previous study reported that the down-regulation of microRNA-195 (miR-195) promotes amyloidogenesis via regulation of amyloid precursor protein and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) expression at the post-transcriptional level in CBH rats with bilateral common carotid artery occlusion (2VO). CBH owing to unilateral common carotid artery occlusion (UCCAO) increases tau phosphorylation levels at multiple phosphorylation sites in the brain, but the molecular mechanism is poorly understood. The purpose of this study was to investigate whether miR-195 could both deregulate amyloid metabolism and indirectly deregulate tau phosphorylation in CBH. We observed that 2VO leads to tau hyperphosphorylation at Ser202/Thr205, Ser262, Thr231, and Ser422 and to the conversion from cyclin-dependent kinase 5 (Cdk5)/p35 to Cdk5/p25 in rat hippocampi. Endogenous miR-195 was knocked down using over-expression of its antisense molecule (pre-AMO-miR-195) via a lentivirus (lenti-pre-AMO-miR-195); this knockdown increased the tau phosphorylation at Ser202/Thr205, Ser262, Thr231, Ser422, and the Cdk5/p25 activation, but over-expression of miR-195 using lenti-pre-miR-195 decreased the tau phosphorylation and Cdk5/p25 activation. Further in vitro studies demonstrated that miR-195 over-expression prevented tau hyperphosphorylation and Cdk5/p35 activity, which were increased by miR-195 inhibition. A dual luciferase reporter assay showed that miR-195 bound to the Cdk5r1 gene, which encodes p35 protein, in the 3'UTR and inhibited p35 expression. We concluded that tau hyperphosphorylation involves the down-regulation of miR-195, which is mediated by Cdk5/p25 activation in 2VO rats. Our findings demonstrated that down-regulation of miR-195 led to increased vulnerability via the regulation of multiple targets. Schematic diagram of miR-195 mediated Aß aggregation and tau hyperphosphorylation in chronic brain hypoperfusion (CBH). First, CBH results in the elevation of nuclear factor-κB (NF-κB), which binds with the promoter sequences of miR-195 and negatively regulates the expression of miR-195. Second, down-regulated miR-195 induces up-regulation of APP and BACE1 and leads to an increase in Aß levels. Third, some of the elevated Aß then enter the intracellular space and activate calpain, which promotes the conversion of Cdk5/p35 to Cdk5/p25 and catalyzes the degradation of IκB; IκB is an inhibitor of NF-κB, which activates NF-κB. Cdk5/p25 directly phosphorylates Tau. Fourth, down-regulated miR-195 induces an up-regulation of p35, which provides the active substrates of p25. Our findings demonstrated that the down-regulation of miR-195 plays a key role in the increased vulnerability to dementia via the regulation of multiple targets following CBH.

Effect of combined treatment with rosuvastatin and protein kinase Cß2 inhibitor on angiogenesis following myocardial infarction in diabetic rats.

The aim of the present study was to investigate the effects of combined treatment with rosuvastatin and LY333531, a selective protein kinase C (PKC)ß2 inhibitor, on angiogenesis under hyperglycemic conditions. Human umbilical vein endothelial cells (HUVECs) cultured in medium containing a normal or high concentration of glucose (33.3 mmol/l) were treated with rosuvastatin (0.1 µmol/l) alone or in combination with LY333531 (10 nmol/l). HUVEC migration and tube formation were assessed. Furthermore, rats with streptozotocin-induced diabetes were randomly divided into groups and treated with either rosuvastatin alone (5 mg/kg/day) or in combination with LY333531 (10 mg/kg/day) for 4 weeks following the induction of myocardial infarction (MI). Echocardiographic patterns, the extent of myocardial fibrosis, capillary density in myocardial tissue, the phosphorylation of Akt and endothelial nitric oxide synthase (eNOS), as well as the expression levels of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor 1-α (HIF­1α) were assessed. The results from the in vitro experiment revealed that the tube-forming and migration ability of the HUVECs exposed to high-glucose medium was significantly improved in the group treated with the combination of rosuvastatin and LY333531. In vivo, the combination of rosuvastatin and LY333531 significantly improved left ventricular function, reduced the extent of myocardial fibrosis and increased myocardial capillary density compared to treatment with rosuvastatin alone. In addition, the expression levels of VEGF, and Akt and eNOS phosphorylation were significantly higher in the group exposed to the combination treatment than in the group treated with rosuvastatin alone. The results of the present study indicate that, compared to treatment with rosuvastatin alone, combined treatment with rosuvastatin and LY333531 promotes a greater level of angiogenesis in diabetic rats with MI. This effect is likely mediated through the upregulation of the VEGF­dependent Akt/eNOS signaling pathway.