Objective: The aim of the present study was to investigate whether pterostilbene could modulate the TLR4/NF-kappa B signaling, reduce neutrophil accumulation and TNF-alpha induction in an ischemia/reperfusion injured rat heart model. Materials and Methods: Rats were randomly exposed to sham operation, myocardial ischemia and reperfusion (MI/R), MI/R + pterostilbene, MI/R + pterostilbene + L-NAME. And myocardial infarct size, apoptosis, TLR4 expression, NF-kappa B expression, MPO level and TNF-alpha level were detected. Results: The results demonstrated that after MI/R, the expressions of myocardial TLR4, NF-kappa B and caspase-3 increased significantly in ischemia area. Compared with MI/R, pterostilbene significantly attenuated the expressions of TLR4, NF-kappa B and caspase-3. In addition, it also reduced myeloperoxidase (MPO) levels, both serum and myocardial TNF-alpha production, myocardial infarct sizes (INF/AAR%) and myocardial apoptosis induced by MI/R. All the effects of pterostilbene were abolished by L-NAME, a nitric oxide synthase inhibitor. Conclusion: These data provide evidence that pterostilbene inhibits TLR4/NF-kappa B signaling and apoptosis in the rat heart subjected to MI/R, which is associated with NO production.

OBJECTIVE To detect the target degradation of miR-141-3p on androgen receptor (AR) gene in LNCaP cells and demonstrate whether AR gene is a target of miR-141-3p. METHODS After prostate cancer cell line LNCaP was transfected with miR-141-3p mimics, expression levels of AR mRNA and protein in the LNCaP cells were detected by reverse transcription PCR and Western blotting, respectively. The 3'untranslated regions (3'UTR) of AR mRNA containing the binding site of miR-141-3p was amplified by PCR and inserted into pmiR-report vector (a 3'downstream luciferase reporter gene). The product was termed pmiR-AR-3'UTR. Double luciferase reporter system was employed to verify the potential target effect of miR-141-3p on pmiR-AR-3'UTR. RESULTS Transfection of miR-141-3p mimics decreased both mRNA and protein expression levels of AR in LNCaP cells. Compared with control group, miR-141-3p transfection significantly inhibited the activity of luciferase of pmiR-AR-3'UTR. CONCLUSION AR is a direct target gene of miR-141-3p.

OBJECTIVE:The aim of the present study was to investigate whether pterostilbene could modulate the TLR4/NF-κB signaling, reduce neutrophil accumulation and TNF-α induction in an ischemia/reperfusion injured rat heart model.MATERIALS AND METHODS:Rats were randomly exposed to sham operation, myocardial ischemia and reperfusion (MI/R), MI/R + pterostilbene, MI/R + pterostilbene + L-NAME. And myocardial infarct size, apoptosis, TLR4 expression, NF-κB expression, MPO level and TNF-α level were detected.RESULTS:The results demonstrated that after MI/R, the expressions of myocardial TLR4, NF-κB and caspase-3 increased significantly in ischemia area. Compared with MI/R, pterostilbene significantly attenuated the expressions of TLR4, NF-κB and caspase-3. In addition, it also reduced myeloperoxidase (MPO) levels, both serum and myocardial TNF-α production, myocardial infarct sizes (INF/AAR%) and myocardial apoptosis induced by MI/R. All the effects of pterostilbene were abolished by L-NAME, a nitric oxide synthase inhibitor.CONCLUSION:These data provide evidence that pterostilbene inhibits TLR4/NF-κB signaling and apoptosis in the rat heart subjected to MI/R, which is associated with NO production.

OBJECTIVE To investigate the cerebral infarct volume 24 hours after transient middle cerebral artery occlusion (tMCAO) and the proportion of CD4⁺;CD25⁺;Foxp3⁺; regulatory T cells (Tregs) in splenocytes in diverse periods after all-trans retinoic acid (ATRA) treatment in mice, so as to explore whether ATRA have the protection against cerebral ischemia damage in mice through intervening Treg differentiation. METHODS Sixty male Kunming mice were randomly divided into two groups, i.e. pretreatment (n=40) and post-treatment (n=20) groups. Each group was against divided into two subgroups, i.e. tMCAO combined with ATRA treatment group, tMCAO combined with DMSO control group. Pretreatment groups: mice were treated intraperitoneally with ATRA (10 mg/kg) dissolved in 100 mL/L DMSO or equivalent volume of 100 mL/L DMSO daily for 7 days (n=20/group). Ten mice in each group were sacrificed and the proportion of Tregs in splenocytes was analyzed by flow cytometry (FCM) after 7-day pretreatment. The other 10 mice in each group were subjected to tMCAO by modified monofilament method. Neurologic deficit score (NDS) was recorded and the infarct volume was assessed by 2, 3, 5-triphenyltetrazolium chloride(TTC) staining 24 hours after tMCAO. The mice in post-treatment groups were treated intraperitoneally with ATRA (10 mg/kg) or equivalent volume of 100 mL/L DMSO immediately after the reperfusion of tMCAO modeling (n=10/group). NDS and infarct volume were assessed and the proportion of Tregs in splenocytes was analyzed 24 hours after tMCAO. RESULTS ATRA pretreatment for 7 days failed to improve neurologic function deficit (P>0.05) and to reduce the cerebral infarct volume (P>0.05) 24 hours after tMCAO in mice. ATRA post-treatment could markedly improve neurologic function (P<0.05) and reduce the cerebral infarct volume (P<0.05) 24 hours after tMCAO. However, neither ATRA pretreatment nor post-treatment had effect on the proportion of Tregs in the splenocytes of mice (P>0.05). CONCLUSION ATRA administered before tMCAO for 7 days failed to protect brain against ischemic damage. ATRA administered immediately following tMCAO induced cerebral protective effect 24 hours after tMCAO. The results suggest that Tregs change is not involved in the neuroprotection mechanism of ATRA.

BACKGROUND: Delayed emergence from general anesthesia frequently occurs in elderly patients, but the reason is not clear. Orexin has been shown to be involved in arousal from general anesthesia. In this study, we examined plasma orexin-A levels in both elderly and young patients during the anesthesia arousal cycle. METHODS: We recruited 41 patients scheduled for elective lumbar surgery and eventually evaluated 34 patients. Patients were divided into a young group (age 30–55, N = 16) and an elderly group (age 65–77, N = 18). Anesthesia with sevoflurane-remifentanil was titrated to maintain the Bispectral Index between 45 and 65. The times from stopping anesthesia to eyes opening and extubation were recorded. Arterial blood was collected, and plasma orexin-A was determined by radioimmunoassay at the following 4 time points: preanesthesia (T0), 1 hour after anesthesia induction (T1), emergence (5 minutes after tracheal extubation) (T2), and 30 minutes after tracheal extubation (T3). RESULTS: The times from stopping anesthesia to eyes opening and tracheal extubation were both significantly longer in the elderly group than in the young group (P = 0.004, P = 0.01, respectively). Basal (T0) orexin-A levels were higher in the elderly group than in the young group (T0, 26.13 ± 1.25 vs 17.9 ± 1.30 pg/mL, P < 0.0001). Plasma orexin-A levels did not change during induction of anesthesia in either group but significantly increased at T2 (vs T0, P <0.0001) in both elderly (35.0 ± 1.7 pg/mL) and young (29.2 ± 1.9 pg/mL) groups. Orexin-A levels were significantly higher in the elderly than in the young group at T1, T2, and T3. CONCLUSION: Plasma orexin-A levels are not responsible for the delayed emergence from general anesthesia in elderly patients.

OBJECTIVES:To investigate the effect of air-drying pressure during ceramic primer coating on zirconia/resin bonding and the surface characteristics of the primed zirconia. METHODS:Two ceramic primers (Clearfil Ceramic Primer, CCP, Kuraray Medical Inc. and Z-Prime Plus, ZPP, Bisco Inc.) were applied on the surface of air-abraded zirconia (Katana zirconia, Noritake) and dried at 4 different air pressures (0.1-0.4 MPa). The primed zirconia ceramic specimens were bonded with a resin-based luting agent (SA Luting Cement, Kuraray). Micro-shear bond strengths of the bonded specimens were tested after 3 days of water storage or 5,000× thermocycling (n = 12). Failure modes of the fractured specimens were examined with scanning electron miscopy. The effects of air pressure on the thickness of the primer layers and the surface roughness (Sa) of primed zirconia were evaluated using spectroscopic ellipsometry (n = 6), optical profilometry and environmental scanning electron microscopy (ESEM) (n = 6), respectively. RESULTS:Clearfil Ceramic Primer air-dried at 0.3 and 0.4 MPa, yielding significantly higher µSBS than gentle air-drying subgroups (p < 0.05). Compared to vigorous drying conditions, Z-Prime Plus air-dried at 0.2 MPa exhibited significantly higher µSBS (p < 0.05). Increasing air-drying pressure reduced the film thickness for both primers. Profilometry measurements and ESEM showed rougher surfaces in the high pressure subgroups of CCP and intermediate pressure subgroup of ZPP. CONCLUSION:Air-drying pressure influences resin/zirconia bond strength and durability significantly. Higher air-drying pressure (0.3-0.4 MPa) for CCP and intermediate pressure (0.2 MPa) for ZPP are recommended to produce strong, durable bonds between resin cement and zirconia ceramics.

AIM:To investigate the efficacy and feasibility of percutaneous intramyocardial injection of bone marrow mesenchymal stem cells (MSC) and autologous bone marrow-derived mononuclear cells (BMMNC) on cardiac functional improvement in porcine myocardial infarcted hearts.METHODS AND RESULTS:Acute myocardial infarction (AMI) was induced in 22 minipigs by temporary balloon occlusion of the left anterior descending coronary artery for 60 min.Two weeks post AMI, BMMNC (n = 7, 245 ± 98 × 10(6)), MSC (n = 8, 56 ± 17 × 10(6)), or phosphate buffered saline (PBS; n = 7) were injected intramyocardially. Cardiac function and myocardial perfusion were analyzed by echocardiography and gated single-photon emission computed tomography/computed tomography (SPECT/CT) at 1 week before AMI and 2 and 10 weeks after AMI. Cell engraftment, proliferation, vascular density, and cardiac fibrosis were evaluated by histology analysis. In all groups, the echocardiography revealed no significant change in the left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV), or left ventricular end-diastolic volume (LVEDV) at 10 weeks after AMI compared with those at 2 weeks after AMI. However, the wall motion score index (WMSI) and left ventricular systolic wall thickening (WT%) were significantly improved at 10 weeks compared with those at 2 weeks after AMI in the MSC group (WMSI 1.55 ± 0.06 vs. 1.87 ± 0.10, WT 33.4 ± 2.3% vs.24.8 ± 2.7%,p < 0.05) but not in the BMMNC group. In addition, myocardial perfusion quantified by SPECT/CT was improved in both the MSC and BMMNC groups, whereas the MSC group showed a superior improvement in vascular density and collagen volume fraction (p < 0.05).CONCLUSION:This preclinically relevant study suggests that when delivered by percutaneous (transcatheter) intramyocardial injection, MSC might be more effective than BMMNC to improve ischemia and reperfusion after AMI.

Aim The aim of the present study was to investigate the role of Lin28a in protecting against hypoxia/reoxygenation (H/R)-induced cardiomyocytes apoptosis under high glucose/high fat (HG/HF) conditions. Methods Primary cardiomyocytes which were isolated from neonatal mouse were randomized to be treated with lentivirus carrying Lin28a siRNA, Lin28acDNA 72 h before H/R (9 h/2 h). Cardiomyocytes biomarkers release (LDH and CK), cardiomyocytes apoptosis, mitochondria biogenesis and morphology, intracellular reactive oxygen species (ROS) production, ATP content and inflammatory cytokines levels after H/R injury in high glucose/high fat conditions were compared between groups. The target proteins of Lin28a were examined by western blot analysis. Results Our results revealed that Lin28a cDNA transfection (overexpression) significantly inhibited cardiomyocyte apoptotic index, improved mitochondria biogenesis, increased ATP production and reduced ROS production as compared with the H/R group in HG/HF conditions. Lin28a siRNA transfection (knockdown) rendered the cardiomyocytes more susceptible to H/R injury as evidenced by increased apoptotic index, impaired mitochondrial biogenesis, decreased ATP production and increased ROS level. Interestingly, these effects of Lin28a were blocked by pretreatment with the PI3K inhibitor wortmannin. Lin28a overexpression increased, while Lin28a knockdown inhibited IGF1R, Nrf-1, Tfam, p-IRS-1, p-Akt, p-mTOR, p-p70s6k, p-AMPK expression levels after H/R injury in HG/HF conditions. Moreover, pretreatment with wortmannin abolished the effects of Lin28a on the expression levels of p-AKT, p-mTOR, p-p70s6k, p-AMPK. Conclusions The present results suggest that Lin28a inhibits cardiomyocytes apoptosis by enhancing mitochondrial biogenesis and function under high glucose/high fat conditions. The mechanism responsible for the effects of Lin28a is associated with the PI3K/Akt dependent pathway.

Objectives Diabetic presents higher ischaemic heart disease morbidity and mortality, lacking of effective strategies of prevention and cure. Recently, a small peptide, helix B surface peptide (HBSP), derived from erythropoietin (EPO) was developed and shown to be tissue protective but avoiding property of elevating red blood cell which might become additional cardiovascular risks for already vulnerable patients. Our preliminary experiment results proved that HBSP could dramatically reduce the myocardial infarction size induced by ischaemia/reperfusion injury (I/RI) of Zucker diabetic fatty rat and the protective effect was superior to EPO. Nevertheless, the specific cardio-protective mechanisms of HBSP still unknown. Purpose of this study was to future elucidate the cardio-protective mechanisms of HBSP against I/RI of diabetic rats. Methods Adult Zucker diabetic fatty rat hearts were subjected 30 min hearts ischaemia and 4h reperfusion. The hearts were randomized to receive EPO-derived peptides HBSP, EPO, HBSP plusLY294002 (specific inhibitor of PI3K), HBSP plus PD98059 (specific inhibitor ofERK1/2) or HBSP plus AG-490 (specific inhibitor of JAK2) at the start of reperfusion. To further confirm the correlation between survivin and myocardial survival in vitro and in vivo, cardiomyocytes were infected with adenovirusencoding survivin or transfected with siRNA targeting survivin. Phosphorylation of Akt, ERK1/2, STAT3 and the expression of protein survivin were determined by Western Blotting and myocardial infarction/area at risk was assessed using TTC staining, cardiomyocytes apoptosis were evaluated after reperfusion by TUNEL staining. Results A single bolus injection of 60 μg/kg of HBSP before reperfusion reduced myocardial infarction size examined 4 h after reperfusion, by (33 ± 5.2)% and apoptosis of cardiomyocytes by (46 ± 2.5)% which was superior to that of EPO (P<0.05) and plus no RBC increasing. HBSP treatment enhanced phosphorylation of all three critical signaling pathways of cell survival, including Akt, ERK1/2, and STAT3 respectively (P<0.05 vs. control). Meanwhile HBSP reperfusion resulted in a4.1-fold increase in survivin expression of myocardium (P<0.05 vs. I/Ralone), which was almost completely blocked by LY294002. Moreover, over-expressed survivin provides protection against simulate dischaemia/reperfusion-induced cardiomyocyte apoptosis, while targeting survivin blunted the anti-apoptotic effect of HBSP. Conclusions This study demonstrates that HBSP up-regulates myocardial survivin expression, mainlyvia PI3K/Akt mechanism, which contributes to the anti-apoptotic effect of HBSPagainst the myocardial I/RI of diabetic rats.

Impaired cardiac microvascular function contributes to cardiovascular complications in diabetes. Glucagon-like peptide-1 (GLP-1) exhibits potential cardioprotective properties in addition to its glucose-lowering effect. This study was designed to evaluate the impact of GLP-1 on cardiac microvascular injury in diabetes and the underlying mechanism involved. Experimental diabetes was induced using streptozotocin in rats. Cohorts of diabetic rats received a 12-week treatment of vildagliptin (dipeptidyl peptidase-4 inhibitor) or exenatide (GLP-1 analog). Experimental diabetes attenuated cardiac function, glucose uptake, and microvascular barrier function, which were significantly improved by vildagliptin or exenatide treatment. Cardiac microvascular endothelial cells (CMECs) were isolated and cultured in normal or high glucose medium with or without GLP-1. GLP-1 decreased high-glucose–induced reactive oxygen species production and apoptotic index, as well as the levels of NADPH oxidase such as p47 and gp91. Furthermore, cAMP/PKA (cAMP-dependent protein kinase activity) was increased and Rho-expression was decreased in high-glucose–induced CMECs after GLP-1 treatment. In conclusion, GLP-1 could protect the cardiac microvessels against oxidative stress, apoptosis, and the resultant microvascular barrier dysfunction in diabetes, which may contribute to the improvement of cardiac function and cardiac glucose metabolism in diabetes. The protective effects of GLP-1 are dependent on downstream inhibition of Rho through a cAMP/PKA-mediated pathway. Diabetes 62:1697–1708, 2013

BACKGROUND Our previous papers indicate that flurbiprofen axetil (FA), a cyclooxygenase inhibitor, is a promising therapeutic strategy for cerebral ischemia in rats. This study aimed to investigate whether FA could promote a neuroprotective effect by activation of peroxisome proliferator-activated receptor-γ (PPAR-γ) after focal cerebral ischemia in rats. METHODS Totally 48 male Sprague-Dawley (SD) rats were randomly assigned into six groups (n = 8 in each group): animals in group ischemia/reperfusion (I/R) only received 120-minute transient middle cerebral artery occlusion (tMCAO); animals in group I/R + FA were administered FA (10 mg/kg) by caudal vein just after 120-minute tMCAO; animals in group I/R + FA + GW9662 were administered GW9662 (a PPAR-γ inhibitor, 1 mg/kg) intraperitoneally 30 minutes before cerebral ischemia onset and FA (10 mg/kg) by caudal vein just after 120-minute tMCAO; animals in group I/R + GW9662 were administered GW9662 (1 mg/kg) intraperitoneally 30 minutes before cerebral ischemia onset; animals in group I/R + DMSO were administered 3% DMSO (vehicle of GW9662, 1 ml/kg) intraperitoneally 30 minutes before cerebral ischemia onset; animals in sham group experienced the identical surgery apart from the insertion of the nylon filament. The neurologic deficit score (NDS) were performed at 72 hours after reperfusion, and then mean brain infarct volume percentage (MBIVP) was determined with 2,3,5-triphenyltetrazolium chloride (TTC) 10 g/L staining. RESULTS NDS was significantly increased in group I/R + FA (12.0 (10.0 - 15.0)), group I/R + FA + GW9662 (10.0 (8.0 - 12.0)), and in group I/R + FA + DMSO (12.0 (9.0 - 14.0)) at 72 hours after reperfusion compared with those in group I/R (7.5 (6.0 - 10.0)). NDS was conspicuously different between group I/R + FA (12.0 (10.0 - 15.0)) and group I/R + FA + GW9662 (10.0 (8.0 - 12.0)). MBIVP in group I/R ((45.82 - 8.83)%) was significantly greater than that in group I/R + FA ((23.52 - 9.90)%), group I/R + FA + GW9662 ((33.17 - 7.15)%); MBIVP in group I/R + FA ((23.52 - 9.90)%) was significantly smaller than that in group I/R + FA + GW9662 ((33.17 - 7.15)%). CONCLUSIONS FA confers the neuroprotective effect on tMCAO in rats and the selective PPAR-γ antagonist GW9662 attenuates the effect of FA. FA could promote a neuroprotective effect by, or in part, activation of PPAR-γ after focal cerebral ischemia in rats.