Hydrogen­rich saline promotes neuronal recovery in mice with cerebral ischemia through the AMPK/mTOR signal­mediated autophagy pathway.

This study explored the protective effect and mechanism of hydrogen­rich saline (HRS) on the neurological function of mice with cerebral ischemia. Effects of HRS on neurological function in mice with cerebral ischemia were evaluated by neurological function scores. Infarct volume and histological damage were evaluated by 2,3,5­triphenyl tetrazolium chloride staining (TTC staining). Golgi­Cox staining was conducted to measure the morphological changes of neuronal dendrites and dendritic spines. The expression of neuronal markers was detected by immunofluorescence. Western blot was used to detect protein expression. The infarct volume of mice in the HRS­H group decreased significantly compared to that of the distal middle cerebral artery occlusion (dMCAO) group. Mice in the HRS­H group had a lower neurological deficit score than that in the dMCAO group. Compared to the dMCAO group, the activity of superoxide dismutase (SOD) and the level of glutathione (GSH) significantly increased in the HRS­H group. Compared with the dMCAO group, the number of apoptotic cells in the HRS­H group decreased. Administration of HRS was shown to be able to decrease cavitation of the brain cortex after ischemia. The spine density in the HRS­H group increased compared to that of the dMCAO group. In the in vitro experiment, compared with the oxygen­glucose deprivation (OGD) group, the active oxygen content in the 75% HRM group decreased, and the mitochondrial membrane potential and adenosine triphosphate (ATP) content increased. Compared with the OGD group, the ratio of P­AMPK and the levels of LC3II/LC3I in the hydrogen­rich medium (HRM) group was upregulated, and P­mTOR levels and P62 levels in the HRM group were down­regulated. HRS can enhance neuroplasticity after ischemia and promote neurological recovery in mice with cerebral ischemia, which may involve the autophagy pathway mediated by the AMPK/mTOR signaling pathway.

Neuroprotective mechanism of salvianolic acid B against cerebral ischemia-reperfusion injury in mice through downregulation of TLR4, p-p38MAPK, p-JNK, NF-κB, and IL-1ß.

OBJECTIVE: Tissue injury and inflammation are two potential outcomes of cerebral ischemia-reperfusion (I/R) injury. Salvianolic acid B (Sal B), isolated from the roots of Salvia miltiorrhiza, is one of the major water-soluble compounds with a wide range of pharmacological effects including antioxidant, anti-inflammatory, antiproliferative, and neuroprotective effects. In the present study, we explored the neuroprotective effects and potential mechanisms of Sal B after I/R injury. METHODS: We induced cerebral ischemia in male CD-1 mice through transient (60 min) middle cerebral artery occlusion (tMCAO), and then injected Sal B (30 mg/kg) intraperitoneally. Neurological deficits, infarct volumes, and brain edema were assessed at 24 and 72 h after tMCAO. We detected the expression of Toll-like receptor 4 (TLR4), phosphorylated-p38 mitogen-activated protein kinase (P-p38 MAPK), phosphorylated c-Jun amino (N)-terminal kinases (p-JNK), nuclear factor-κB (NF-κB), and interleukin-1ß (IL-1ß) in the brain tissue. RESULTS: Compared with the tMCAO group, Sal B significantly improved neurological deficits, reduced infarct size, attenuated cerebral edema, and downregulated the expression of pro-inflammatory mediators TLR4, p-p38MAPK, p-JNK, nuclear NF-κB, and IL-1ß in brain tissue after I/R injury. CONCLUSION: We found that Sal B protects brain tissues from I/R injury by activating its anti-inflammatory properties.

Indocyanine green combined with methylene blue versus methylene blue alone for sentinel lymph node biopsy in breast cancer: a retrospective study.

BACKGROUND: Recent studies have shown that near-infrared (NIR) fluorescence imaging using Indocyanine green (ICG) may improve the efficiency of sentinel lymph node biopsy (SLNB). This study aimed to assess the effectiveness of the combination of ICG and methylene blue (MB) in breast cancer patients undergoing SLNB. PATIENTS AND METHOD: We evaluated ICG plus MB (ICG + MB) identification effectiveness with MB alone using retrospective analysis. From 2016 to 2020, we collected data on 300 eligible breast cancer patients who got SLNB treatment in our institution by ICG + MB or MB alone. By comparing the distribution of clinicopathological characteristics, the detection rate of sentinel lymph nodes (SLNs) and metastatic SLNs, as well as the total number of SLNs in the two groups, we were able to assess the imaging efficiency. RESULTS: Fluorescence imaging allowed 131 out of 136 patients in the ICG + MB group to find SLNs. ICG + MB group and MB group had detection rates of 98.5% and 91.5% (P = 0.007, χ2 = 7.352), respectively. Besides, the ICG + MB approach was able to produce improved recognition outcomes. What's more, compared with the MB group, the ICG + MB group can identify more lymph nodes (LNs) (3.1 to 2.6, P = 0.000, t = 4.447). Additionally, in the ICG + MB group, ICG could identify more LNs than MB (3.1 vs 2.6, P = 0.004, t = 2.884). CONCLUSION: ICG has high detection effectiveness for SLNs, and when paired with MB, the detection efficiency can be increased even further. Furthermore, the ICG + MB tracing mode does not involve radioisotopes, which has a lot of promise for clinical use and can take the place of conventional standard detection methods.

Adenosine mono-phosphate-activated protein kinase-mammalian target of rapamycin signaling participates in the protective effect of chronic intermittent hypobaric hypoxia on vascular endothelium of metabolic syndrome rats.

Our previous study demonstrated that chronic intermittent hypobaric hypoxia (CIHH) protects vascular endothelium function through ameliorating autophagy in mesenteric arteries of metabolic syndrome (MS) rats. This study aimed to investigate the role of adenosine mono-phosphate-activated protein kinase-mammalian target of rapamycin (AMPK-mTOR) signaling in CIHH effect. Six-week-old male Sprague-Dawley rats were divided into control (CON), MS model, CIHH treatment (CIHH), and MS + CIHH groups. Serum pro-inflammatory cytokines were measured. The endothelium dependent relaxation (EDR), endothelial ultrastructure and autophagosomes were observed in mesenteric arteries. The expression of phosphor (p)-AMPKα, p-mTOR, autophagy-related and endoplasmic reticulum stress-related proteins, p-endothelial nitric oxide synthase, and cathepsin D were assayed. In MS rats, pro-inflammatory cytokines were increased, EDR was attenuated, and endothelial integrity was impaired. In addition, the expression level of p-AMPKα and cathepsin D was down-regulated, but the level of p-mTOR was up-regulated. While in MS + CIHH rats, all aforementioned abnormalities were ameliorated, and the beneficial effect of CIHH was cancelled by AMPKα inhibitor. In conclusion, AMPK-mTOR signaling pathway participates in the protection of CIHH on vascular endothelium of MS rats.

CIHH protects the heart against left ventricular remodelling and myocardial fibrosis by balancing the renin-angiotensin system in SHR.

AIM: The aim of our study was to clarify the cardioprotection of chronic intermittent hypobaric hypoxia (CIHH) and the underlying mechanism in spontaneously hypertensive rats (SHR). MAIN METHODS: Adult male rats were divided into normal blood pressure Wistar-Kyoto rats (WKY) control (WKY-CON), WKY rats with CIHH treatment (WKY-CIHH), SHR control (SHR-CON) and SHR with CIHH treatment (SHR-CIHH) groups. SHR-CIHH and WKY-CIHH rats were subjected to hypobaric hypoxia simulating 4000-m altitude for 35 days, 5 h per day. Arterial blood pressure and cardiac function parameters, including ejection fraction, fractional shortening and left ventricular (LV) wall thickness, were evaluated. Cardiac pathomorphology and myocardial fibrosis were determined. The expression of angiotensin-converting enzyme (ACE), ACE2, Ang II, Ang1-7, AT1 receptor, Mas receptor, IL-6, TNF-α，IL-10, SOD and MDA were assayed in myocardium. KEY FINDINGS: CIHH significantly decreased arterial blood pressure, alleviated LV hypertrophy, and improved cardiovascular function in SHR (P < 0.05-0.01). Also, CIHH protected SHR heart against morphological changes and fibrosis. In addition, CIHH significantly down-regulated the ACE/Ang II/AT1 receptor axis and up-regulated the ACE2/Ang1-7/Mas axis of renin-angiotensin system (RAS) in SHR (P < 0.05-0.01). CIHH significantly reduced IL-6, TNF-α, and MDA levels, but increased IL-10 and SOD in SHR myocardium (P < 0.05-0.01). SIGNIFICANCE: The CIHH treatment protected the heart of SHR against LV remodelling and myocardial fibrosis, which might be carried out through a balance in the ACE/Ang II/AT1 axis and the ACE2/Ang1-7/Mas axis of the RAS to reduce inflammation, and inhibit oxidative stress.

Elevated expression of the leptin receptor ob­R may contribute to inflammation in patients with ulcerative colitis.

The effect of leptin on ulcerative colitis (UC) has been controversial. The present study aimed to investigate the role of leptin and its receptor ob­R in UC and the underlying mechanism of this role. The level of serum leptin and the protein expression of the leptin receptor ob­R in the colonic mucosa were determined in patients with UC. Experimental colitis was induced through intrarectal administration of 2,4,6­trinitrobenzene sulfonic acid (TNBS) in leptin receptor­deficient Zucker rats (LR­D). The body weight, disease activity index, colon length, and macroscopic and histopathological appearance were evaluated. Furthermore, the myeloperoxidase (MPO) enzyme activity and cytokine levels in colon tissues were also determined. The expression of the signal transducer and activator of transcription 3 (STAT3), phosphorylated STAT3 (p­STAT3), nuclear factor (NF)­κB­p65, and Ras homolog gene family member A (RhoA) proteins in colon tissues was assessed. The results revealed that the expression of the leptin receptor ob­R was increased in the colonic mucosa but the serum leptin level was not altered in patients with UC compared with healthy volunteers. The severity of experimental colitis, represented by body weight loss, disease activity index, colon length, and macroscopic and histological changes, was ameliorated in LR­D rats compared with the wild­type (WT) rats. Moreover, the MPO activity; levels of cytokines including interleukin (IL)­1ß, IL­6, and tumor necrosis factor­α; and expression of p­STAT3, NF­κB, and RhoA proteins were reduced in colon tissues of LR­D rats compared with WT rats. In conclusion, activation of the leptin receptor ob­R is an important pathogenic mechanism of UC, and leptin receptor deficiency may provide resistance against TNBS­induced colitis by inhibiting the NF­κB and RhoA signaling pathways.

Protective effects of leonurine against ischemic stroke in mice by activating nuclear factor erythroid 2-related factor 2 pathway.

AIMS: Leonurine has been shown to trigger antioxidant responses during ischemic stroke, and nuclear factor erythroid 2-related factor 2 (Nrf-2) imparts protective effects against oxidative injury. The present study has determined that leonurine prevents ischemic injury of brain tissues via Nrf-2 pathway activation. METHODS: Male ICR mice and Nrf-2-/- mice were subjected to permanent middle cerebral artery occlusion (pMCAO) and received leonurine treatment at 2 hours after pMCAO by intraperitoneal injection. Neurological deficit scores as well as infarct volume were assessed to determine the neuroprotective role of leonurine. Nrf-2 was investigated using Western blotting and real-time polymerase chain reaction (RT-PCR) analysis to elucidate the neuroprotective mechanism of leonurine. Commercial kits were employed to determine reactive oxygen species (ROS), superoxide (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), malonaldehyde (MDA), and glutathione (GSH). Vascular endothelial growth factor (VEGF) was evaluated by Western blotting and RT-PCR analysis, and VEGF was localized using immunofluorescence. RESULTS: The application of leonurine on ICR mice resulted in an improvement in neurological deficit scores and a reduction in infarct volume. Leonurine upregulated nuclear Nrf-2 protein and increased total Nrf-2 protein expression and mRNA levels. Leonurine regulated SOD, MDA, CAT, GSH, and GSH-Px, and it significantly inhibited ROS production in ICR mice. Leonurine improved VEGF expression and increased VEGF expression in neurons, astrocytes, and endothelial cells. However, leonurine had no obvious beneficial effects on Nrf-2-/- mice. CONCLUSIONS: Leonurine exerted neuroprotective effects, promoted antioxidant responses, and upregulated VEGF expression by activating the Nrf-2 pathway.

Rosmarinic acid elicits neuroprotection in ischemic stroke via Nrf2 and heme oxygenase 1 signaling.

Rosmarinic acid (RA) can elicit a neuroprotective effect against ischemic stroke, but the precise molecular mechanism remains poorly understood. In this study, an experimental ischemic stroke model was established in CD-1 mice (Beijing Vital River Laboratory Animal Technology, Beijing, China) by occluding the right middle cerebral artery for 1 hour and allowing reperfusion for 24 hours. After intraperitoneally injecting model mice with 10, 20, or 40 mg/kg RA, functional neurological deficits were evaluated using modified Longa scores. Subsequently, cerebral infarct volume was measured using TTC staining and ischemic brain tissue was examined for cell apoptosis with TUNEL staining. Superoxide dismutase activity and malondialdehyde levels were measured by spectrophometry. Expression of heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), Bcl-2, Bax, Akt, and phospho-Ser473 Akt proteins in ischemic brain tissue was detected by western blot, while mRNA levels of Nrf2, HO-1, Bcl-2, and Bax were analyzed using real time quantitative PCR. In addition, HO-1 enzyme activity was measured spectrophotometrically. RA (20 and 40 mg/kg) greatly improved neurological function, reduced infarct volume, decreased cell apoptosis, upregulated Bcl-2 protein and mRNA expression, downregulated Bax protein and mRNA expression, increased HO-1 and Nrf2 protein and mRNA expression, increased superoxide dismutase activity, and decreased malondialdehyde levels in ischemic brain tissue of model mice. However, intraperitoneal injection of a HO-1 inhibitor (10 mg/kg zinc protoporphyrin IX) reversed the neuroprotective effects of RA on HO-1 enzyme activity and Bcl-2 and Bax protein expression. The PI3K/Akt signaling pathway inhibitor LY294002 (10 mM) inhibited Akt phosphorylation, as well as Nrf2 and HO-1 expression. Our findings suggest that RA has anti-oxidative and anti-apoptotic properties that protect against ischemic stroke by a mechanism involving upregulation of Nrf2 and HO-1 expression via the PI3K/Akt signaling pathway.

Loss of neuronal CD200 contributed to microglial activation after acute cerebral ischemia in mice.

CD200 has been proved to play a role in immuno-inflammatory reaction. However, little information is available on CD200 in the acute stage of cerebral ischemia. We investigated the association between neuronal death and expression of CD200, and explored the relationship between CD200 and microglia in cerebral ischemic mice. Firstly, localization of CD200 expression in the normal brain tissue was detected by immunofluorescent assay. Then, focal cerebral ischemia was induced in mice by permanent middle cerebral artery occlusion (pMCAO) and then cortical tissues were collected at 6, 12, 24 and 48 h after surgery. Changes of CD200 and neuron-specific enolase (NSE) after pMCAO were assessed by western blotting. Meanwhile flow cytometry analysis was implemented to analyze the death of cortical cells. Results of these two parts were analyzed by Pearson correlation analysis. To further study, intracerebroventricular (ICV) injection of recombinant CD200 (rCD200) protein was carried out immediately after pMCAO. Iba-1 was measured by western blotting to evaluate activation of microglia, and inflammatory cytokines including IL-1ß, TNF-α and IL-10 were tested by enzyme-linked immuno sorbent assay (ELISA). The results showed that CD200 was expressed in neurons and was not observed on mircroglia in cortex of normal mice. Expression of CD200 was decreased within 48 h after pMCAO, with a concomitant decrease of NSE expression. The rate of neuronal cell death was approximately around 30% and statistical analysis revealed a negative correlation between level of CD200 and the rate of neuronal death. Compared with control, exogenous rCD200 reduced expressions of Iba-1, IL-1ß, TNF-α and IL-10. Taking together, our results demonstrated that loss of CD200 was caused by neuronal death and was one of contributing factors in microglial activation after cerebral ischemia. ICV injection of rCD200 protein could suppress activation of microglia in vivo.

Chronic intermittent hypobaric hypoxia protects vascular endothelium by ameliorating autophagy in metabolic syndrome rats.

AIMS: The study aimed to investigate the protective effect of chronic intermittent hypobaric hypoxia (CIHH) on endothelium function and relaxation of mesenteric artery in metabolism syndrome (MS) rats. MAIN METHODS: Male adult Sprague-Dawley rats were randomly divided into control (CON), CIHH (treated with 28-days hypobaric hypoxia simulating an altitude of 5000 m, 6 h daily), MS (induced by high fat diet and 10% fructose water feeding), and MS + CIHH groups. Body weight, systolic arterial pressure, blood biochemical and the endothelium dependent relaxation (EDR) of mesenteric arteries were measured. The expression of phosphor-endothelial nitric oxide synthase (p-eNOS), endoplasmic reticulum (ER) stress-related proteins and autophagy-related proteins in mesenteric arteries was assayed. KEY FINDINGS: The MS rats displayed hypertension, obesity, metabolic abnormity and insulin resistance, EDR was attenuated, p-eNOS expression was down-regulated, the expressions of ER stress-related proteins were up-regulated, and autophagy dysfunction occurred. All aforementioned abnormalities in MS rats were ameliorated in MS + CIHH rats. Furthermore, the improvement of CIHH on EDR and p-eNOS was cancelled by the ER stress inducer, and the autophagy inhibitor. SIGNIFICANCE: In conclusion CIHH protects endothelium function and enhances relaxation in mesenteric arteries of MS rats through improving autophagy function, reducing ER stress and up-regulating p-eNOS.

Ulinastatin protects brain against cerebral ischemia/reperfusion injury through inhibiting MMP-9 and alleviating loss of ZO-1 and occludin proteins in mice.

BACKGROUND: The effects of Ulinastatin (UTI) on the blood-brain barrier (BBB) in the acute phase of cerebral ischemia/reperfusion (I/R) are not clear. This study was to investigate the potential protective effects of UTI on the BBB and the underlying mechanisms. METHODS: Male CD-1 mice were subjected to transient middle cerebral artery occlusion (tMCAO) and randomly assigned to four groups: Sham (sham-operated), tMCAO (tMCAO+0.9% saline), UTI-L (tMCAO+UTI 1500U/100g) and UTI-H (tMCAO+UTI 3000U/100g) group. UTI was administered immediately after reperfusion in the UTI-L and UTI-H groups. At 24h after reperfusion, the neurological deficit, brain water content, and infarct volume were determined. Western blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were used to examine the expression of matrix metalloproteinase (MMP)-9, Zonula occludens-1 (ZO-1) and occludin in ischemic cerebral cortex. The integrity of the BBB was assessed by the leakage of Evans blue. RESULTS: Compared with tMCAO group, both UTI-L and UTI-H groups showed significantly (P<0.001) ameliorated the neurological deficit (2.00±0.71 and 1.60±0.55 vs. 4.60±0.55), lessened brain water content (82.99%±0.21% and 82.05%±0.59% vs. 84.28%±0.0.57%) and decreased the infarct volume (38.52%±1.72% and 24.78%±1.20% vs. 49.48%±1.93%). In addition, significantly (P<0.001) decreased expression of MMP-9 (0.48±0.06 and 0.37±0.05 vs.0.76±0.10 for protein and 2.88±0.23 and 2.17±0.16 vs. 3.90±0.24 for mRNA) and alleviated loss of ZO-1 (0.19±0.04 and 0.24±0.05 vs. 0.25±0.03) and occludin (0.74±0.08 and 0.87±0.07 vs. 0.94±0.06) proteins were observed in both UTI-L and UTI-H groups. CONCLUSION: UTI protects the brain against ischemic injury potentially via down-regulating the expression of MMP-9 and alleviating loss of ZO-1 and occludin proteins to restore the BBB permeability.

Limb remote ischemia per-conditioning protects the heart against ischemia-reperfusion injury through the opioid system in rats.

Remote ischemia per-conditioning (RPerC) has been demonstrated to have cardiac protection, but the underlying mechanism remains unclear. This study aimed to investigate the mechanism underlying cardiac protection of RPerC. Adult male Sprague-Dawley rats were used in this study. Cardiac ischemia/reperfusion (I/R) was induced by 30 min of occlusion and 3 h of reperfusion of the left anterior descending coronary artery. RPerC were performed by 5 min of occlusion of the right femoral artery followed by 5 min of reperfusion for three times during cardiac ischemia. The hemodynamics, left ventricular function, arrhythmia, and infarct area were measured. Protein expression levels of endothelial nitric oxide synthase (eNOS), inducible NOS (iNOS), protein kinase C-ε (PKCε), and PKCδ in the myocardium were assayed. During I/R, systolic artery pressure and left ventricular function were decreased, infarct area was increased, and arrhythmia score was increased (P < 0.05). However, changes of the above parameters were significantly attenuated in RPerC-treated rats compared with control rats (P < 0.05). The cardiac protective effects of RPerC were prevented by naloxone or glibenclamide. Also, RPerC increased the protein expression levels of eNOS, iNOS, PKCε, and PKCδ in the myocardium compared with control rats. These effects were blocked by naloxone, an opioid receptor antagonist, and glibenclamide, an ATP-sensitive K+ channel blocker (KATP). In summary, this study suggests that RPerC protects the heart against I/R injury through activation of opioid receptors and the NO-PKC-KATP channel signaling pathways.

Finasteride inhibited brain dopaminergic system and open-field behaviors in adolescent male rats.

AIMS: Finasteride inhibits the conversion of testosterone to dihydrotestosterone. Because androgen regulates dopaminergic system in the brain, it could be hypothesized that finasteride may inhibit dopaminergic system. The present study therefore investigates the effects of finasteride in adolescent and early developmental rats on dopaminergic system, including contents of dopamine and its metabolites (dihydroxy phenyl acetic acid and homovanillic acid) and tyrosine hydroxylase expressions both at gene and protein levels. Meanwhile, open-field behaviors of the rats are examined because of the regulatory effect of dopaminergic system on the behaviors. METHODS: Open-field behaviors were evaluated by exploratory and motor behaviors. Dopamine and its metabolites were assayed by liquid chromatography-mass spectrometry. Tyrosine hydroxylase mRNA and protein expressions were determined by real-time qRT-PCR and western blot, respectively. RESULTS: It was found that in adolescent male rats, administration of finasteride at doses of 25 and 50 mg/kg for 14 days dose dependently inhibited open-field behaviors, reduced contents of dopamine and its metabolites in frontal cortex, hippocampus, caudate putamen, nucleus accumbens, and down-regulated tyrosine hydroxylase mRNA and protein expressions in substantia nigra and ventral tegmental area. However, there was no significant change of these parameters in early developmental rats after finasteride treatment. CONCLUSION: These results suggest that finasteride inhibits dopaminergic system and open-field behaviors in adolescent male rats by inhibiting the conversion of testosterone to dihydrotestosterone, and imply finasteride as a potential therapeutic option for neuropsychiatric disorders associated with hyperactivities of dopaminergic system and androgen.

BRAFV600E mutation is not associated with central lymph node metastasis in all patients with papillary thyroid cancer: Different histological subtypes and preoperative lymph node status should be taken into account.

The association between central lymph node metastasis (LNM) and risk factors, including the presence of the BRAF mutation, BRAFV600E, in patients with papillary thyroid cancer (PTC) requires further investigation. A potent risk factor that can indicate LNM in different histological subtypes of PTC and in different preoperative central lymph node statuses also requires further research. A total of 287 patients with PTC who accepted thyroidectomy were included in the present study. Clinicopathological data of these patients were reviewed to examine the risk factors for central LNM through univariate and multivariate analyses. Overall, BRAFV600E in patients with cN0 (subclinical nodal disease) and cN1 (other than cN0) PTC was associated with central LNM. However, multivariate analyses demonstrated that BRAFV600E was not an independent risk factor in patients with cN1 or cN0 PTC. For patients with classical variant PTC (CVPTC), BRAFV600E was independently associated with central LNM. However, on further analysis, the association was only significant in patients with cN0 CVPTC. For patients with follicular variant PTC (FVPTC) or aggressive variant PTC (AVPTC), the BRAFV600E mutation rate was not significantly different between patients with and without central LNM. In conclusion, BRAFV600E was an independent risk factor for central LNM overall in patients with PTC and in patients with CVPTC, particularly in patients with cN0 CVPTC. However, BRAFV600E was not an independent risk factor for patients with FVPTC and AVPTC. Therefore, BRAFV600E provides varied clinical significance in different histological subtypes and preoperative central lymph node status.

EZH2-mediated α-actin methylation needs lncRNA TUG1, and promotes the cortex cytoskeleton formation in VSMCs.

Recent studies have revealed that long non-coding RNAs (lncRNAs) participate in vascular homeostasis and pathophysiological conditions development. But still very few literatures elucidate the regulatory mechanism of non-coding RNAs in this biological process. Here we identified lncRNA taurine up-regulated gene 1 (TUG1) in rat vascular smooth muscle cells (VSMCs), and got 4612bp nucleotide sequence. The expression level of TUG1 RNA was increased in synthetic VSMCs by real-time PCR analysis. Meanwhile, the expression of enhancer of zeste homolog 2 (EZH2) (TUG1 binding protein) increased in cytoplasm of VSMCs under the same conditions. Immunofluoresce analysis displayed the colocalization of EZH2 with α-actin in cytoplasm and F-actin in cell edge ruffles. This leads us to hypothesize the existence of cytoplasmic TUG1/EZH2/α-actin complex. Using RNA pull down assay, we found that TUG1 interacted with both EZH2 and α-actin. Disruption of TUG1 abolished the interaction of EZH2 with α-actin, and accelerated depolymerization of F-actin in VSMCs. Based on EZH2 methyltransferase activity and the potential methylation sites in α-actin structure, we revealed that α-actin was lysine-methylated. Furthermore, the methylation of α-actin was inhibited by knockdown of TUG1. In conclusion, these findings partly suggested that EZH2-mediated methylation of α-actin may be dependent on TUG1, and thereby promotes cortex F-actin polymerization in synthetic VSMCs.

A scoring system is an effective tool for predicting central lymph node metastasis in papillary thyroid microcarcinoma: a case-control study.

BACKGROUND: The purpose of this study was to evaluate the clinicopathologic and ultrasonographic (US) characteristics and establish an effective scoring system for predicting central lymph node metastasis (CLNM) in papillary thyroid microcarcinoma (PTMC). METHODS: A total of 498 patients with PTMC who underwent total thyroidectomy or lobectomy with therapeutic central lymph node dissection (CLND) were enrolled. Univariate and multivariate analyses were performed to find the independent predictors for CLNM based on clinicopathological and US characteristics. Using the standardized regression coefficient, a 10-point score system was constructed in line with these independent predictors. Then, the scoring system was evaluated for the diagnostic value in predicting CLNM. RESULTS: Tumor location (the lower polo), tumor size (>5 mm), extrathyroidal extension, margin (no well-defined), display of enlarged lymph node, and contact of >25% with the adjacent capsule were independent predictors for CLNM. Verifying the scoring system, a cutoff value of 5 points was found to be the best prediction for CLNM, the sensitivity and specificity were 64.7 and 80.5%, respectively, and the positive and negative predictive values were 77.3 and 69.0%, respectively. CONCLUSIONS: The points≤5 could be considered as a low risk for CLNM, and the points>5 could be identified as a high risk for CLNM. More advanced diagnostic approaches and prophylactic CLND are needed for patients with the points>5.

Apelin-13 protects against apoptosis by activating AMP-activated protein kinase pathway in ischemia stroke.

Apelin has been proved to be protective against apoptosis induced by ischemic reperfusion. However, mechanisms whereby apelin produces neuroprotection remain to be elucidated. AMP-activated protein kinase (AMPK) is a master energy sensor that monitors levels of key energy metabolites. It is activated via AMPKαThr172 phosphorylation during cerebral ischemia and appears to be neuroprotective. In this study, we investigated the effect of apelin on AMPKα and tested whether apelin protecting against apoptosis was associated with AMPK signals. Focal transient cerebral ischemia/reperfusion (I/R) model in male ICR mice was induced by 60 min of ischemia followed by reperfusion. Apelin-13 was injected intracerebroventricularly 15 min before reperfusion. AMPK inhibitor, compound C, was injected to mice intraperitoneally at the onset of ischemia. In experiment 1, the effect of apelin-13 on AMPKα was measured. In experiment 2, the relevance of AMPKα and apelin-13' effect on apoptosis was measured. Data showed that apelin-13 significantly increased AMPKα phosphorylation level after cerebral I/R. Apelin-13, with the co-administration of saline, reduced apoptosis cells, down-regulated Bax and cleaved-caspase3 and up-regulated Bcl2. However, with the co-administration of compound C, apelin-13 was inefficient in affecting apoptosis and Bax, Bcl2 and cleaved-caspase3. The study provided the evidence that apelin-13 up-regulated AMPKα phosphorylation level in cerebral ischemia insults and AMPK signals participated in the mechanism of apelin-mediated neuroprotection.

Electroacupuncture at Qiuxu (GB 40) for treatment of migraine--a clinical multicentral random controlled study.

OBJECTIVE: To observe the therapeutic effects of electroacupuncture (EA) at Qiuxu (GB 40) for treatment of migraine so as to provide clinical evidence for compilation of the Acupoints' Dictionary of the People's Republic of China. METHODS: 275 migraine patients admitted in 3 hospitals were randomly divided into a treatment group treated by EA at Qiuxu (GB 40), and a control group treated by EA at Tianshu (ST 25). The indexes of the migraine symptoms and the 5-HT level were observed in both the groups before and after treatment. RESULTS: There was a significant difference in VAS score between the two groups of the 3 clinical centers (P<0.01). The therapeutic effects of a 4-week treatment were much better in the treatment group than that of the control group. The 3-month follow-up survey showed that the long-term effects were in favor of the treatment group of the first and third clinical centers, though no significant difference was found in the treatment group of the second clinical center as compared with the control group. The results from the 6-month follow-up survey showed better effects in the treatment group of all the 3 clinical centers. CONCLUSION: EA at Qiuxu (GB 40) may show effect for migraine.

Oxymatrine protects rat brains against permanent focal ischemia and downregulates NF-kappaB expression.

BACKGROUND: Oxymatrine is proven to protect ischemic and reperfusion injury in liver, intestine and heart, this effect is via anti-inflammation and anti-apoptosis. Whether this protective effect applies to ischemic injury in brain, we therefore investigate the potential neuroprotective role of oxymatrine and the underlying mechanisms. METHODS: Male, Sprague-Dawley rats were randomly assigned to four groups: permanent middle cerebral artery occlusion (pMCAO), high dose (pMCAO+oxymatrine 120 mg/kg), low dose (pMCAO+oxymatrine 60 mg/kg) and sham operated group. We used a permanent middle cerebral artery occlusion model and administered oxymatrine intraperitoneally immediately after cerebral ischemia and once daily on the following days. At 24 h after MCAO, neurological deficit was evaluated using a modified six point scale; brain water content was measured; NF-kappaB expression was measured by immunohistochemistry, Western blotting and RT-PCR. Infarct volume was analyzed with 2, 3, 5-triphenyltetrazolium chloride (TTC) staining at 72 h. RESULTS: Compared with pMCAO group, neurological deficit in high dose group was improved (P<0.05), infarct volume was decreased (P<0.001) and cerebral edema was alleviated (P<0.05). Consistent with these indices, immunohistochemistry, Western blot and RT-PCR analysis indicated that NF-kappaB expression was significantly decreased in high dose group. Low dose of oxymatrine did not affect NF-kappaB expression in pMCAO rats. CONCLUSIONS: Oxymatrine reduced infarct volume induced by pMCAO, this effect may be through the decreasing of NF-kappaB expression.

Brain damage related to hemorrhagic transformation following cerebral ischemia and the role of K ATP channels.

BACKGROUND: Hemorrhagic transformation (HT) is a major factor limiting the use of thrombolytic treatment for stroke. Animal model can help us to understand HT. This study is to establish a HT model in rats to compare HT with uncomplicated cerebral infarction in neurobehavioral deficit, brain edema, brain adenosine triphosphatase (ATPase) activity and succinic dehydrogenase (SDH) activity, and to investigate its pathology changes as well as the impact of, Glibenclamide, a ATP-sensitive K(+) channel (K(ATP) channel) blocker, on the pathogenesis of HT. METHODS: Male, Sprague-Dawley rats were randomly assigned to four groups: hemorrhagic transformation (HT), cerebral infarction (CI), Glibenclamide+HT (GH) and a control. To create HT model, right middle cerebral artery occlusion (MCAO) was conducted with intraluminal thread technique; 30 min after MCAO, 50 microL arterial blood was injected into the caudate nucleus where the infarction occurred. Neurologic deficit was evaluated by Longa test, Berderson test and Beam test. Brain water content, brain ATPase activity and SDH activity were measured. Histology was examined using light microscope and transmission electron microscope. RESULTS: No significant difference in neurobehavioral deficit and brain water content was observed between HT and CI groups in all time points (P>0.05). Brain ATPase activity 12 h and 24 h after operation and brain SDH activity 24 h after the operation in HT group were both significantly increased compared with those in CI group (P<0.05); the increase of brain ATPase and SDH activity in HT group could be prevented by Glibenclamide. Neuronal degeneration and tissue edema in HT group, swollen neuropil and loosen intercellular substance in CI group were revealed by histology study. Ultrastructural changes including swollen mitochondria and interstitial edema were also observed in both HT and CI groups. CONCLUSIONS: The results demonstrated that moderate hemorrhagic transformation does not significantly aggravate cerebral infarction, and that K(ATP) channels have an important role in energy metabolism.