Cardioprotective effects of luteolin on ischemia/reperfusion injury in diabetic rats are modulated by eNOS and the mitochondrial permeability transition pathway.

Myocardial ischemia/reperfusion (I/R) injury in diabetes is associated with oxidative stress, endothelial nitric oxide synthase (eNOS) dysfunction, and mitochondrial collapse, whereas luteolin is known to protect the cardiovascular system against diabetes and I/R injury. Here, we investigated whether luteolin pretreatment diminishes myocardial I/R injury in diabetic rats by affecting eNOS and the mitochondrial permeability transition pore (mPTP). After diabetic rats were produced by streptozotocin treatment (65 mg/kg) for 3 weeks, luteolin (100 mg·kg·d) or L-NAME (25 mg·kg·d) was administered intragastrically for 2 weeks. Hearts were then isolated and subjected to 30 minutes of global ischemia followed by 120 minutes of reperfusion. Pretreatment with luteolin significantly improved left ventricular function and coronary flow throughout reperfusion, increased cardiac tissue viability and manganese superoxide dismutase (MnSOD) activity, and reduced coronary lactate dehydrogenase release, and the myocardial malonaldehyde level in diabetic I/R rat hearts. All these improving effects of luteolin were significantly attenuated by L-NAME. Luteolin also significantly upregulated eNOS expression in diabetic rat hearts after I/R. Ca-induced mPTP opening and mitochondrial inner membrane potential reduction were significantly inhibited in ventricular myocytes isolated from luteolin-treated diabetic rats, and this effect was attenuated by L-NAME. These findings indicate that luteolin protects the diabetic heart against I/R injury by upregulating the myocardial eNOS pathway, and downstream effects include the enhancement of MnSOD and inhibition of mPTP.

Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain.

BACKGROUND: The analgesic potency of opioids is reduced in neuropathic pain. However, the molecular mechanism is not well understood. RESULTS: The present study demonstrated that increased methylation of the Mu opioid receptor (MOR) gene proximal promoter (PP) in dorsal root ganglion (DRG) plays a crucial role in the decreased morphine analgesia. Subcutaneous (s.c.), intrathecal (i.t.) and intraplantar (i.pl.), not intracerebroventricular (i.c.v.) injection of morphine, the potency of morphine analgesia was significantly reduced in nerve-injured mice compared with control sham-operated mice. After peripheral nerve injury, we observed a decreased expression of MOR protein and mRNA, accompanied by an increased methylation status of MOR gene PP, in DRG. However, peripheral nerve injury could not induce a decreased expression of MOR mRNA in the spinal cord. Treatment with 5-aza-2'-deoxycytidine (5-aza-dC), inhibited the increased methylation of MOR gene PP and prevented the decreased expression of MOR in DRG, thereby improved systemic, spinal and periphery morphine analgesia. CONCLUSIONS: Altogether, our results demonstrate that increased methylation of the MOR gene PP in DRG is required for the decreased morphine analgesia in neuropathic pain.

[Enhancing effects of perlecan or vascular endothelial growth factor-165 on angiogenesis in mice].

OBJECTIVE: To explore the angiogenesis-promoting effects of perlecan or vascular endothelial growth factor (VEGF) at different time points through quantitative analysis of microvessel density (MVD). METHODS: Four kinds of scaffolds: open-cell polylactic acid (OPLA) (control), OPLA + VEGF-165, OPLA + perlecan and OPLA + VEGF-165 + perlecan were implanted into mice. At Weeks 1, 2, 3, 4, 6 and 8, OPLA was harvested and then HE and immunocytochemistry were employed to detect the angiogenesis-promoting effects of scaffold. MVD of each group was appraised by the Tukey test. RESULTS: The scaffolds exhibited excellent biocompatibility with tissue. Numerous vessels were spotted obviously around the implants, especially at Week 8. And the OPLA scaffold degraded with the elapsing of time and its inner part was divided into many sections along with the ingrowth of vessels. Compared with the other three groups, the MVD of the OPLA + VEGF-165 + perlecan group was the highest at all time points. There were statistical differences between the OPLA + VEGF-165 + perlecan and OPLA groups at Week 1 (3.30 ± 0.42 vs 1.80 ± 0.29); MVD of the OPLA + VEGF-165 + perlecan group was thrice as much as the OPLA group at Week 3 (11.70 ± 0.87 vs 4.50 ± 0.34); at Week 8, MVD of the OPLA + VEGF-165 + perlecan group was more than thrice as much as the OPLA group (31.40 ± 1.35 vs 9.90 ± 0.67). CONCLUSION: Angiogenesis is synergistically enhanced by the combined application of VEGF-165 and perlecan in mice.

Sestrin2 is involved in the Nrf2-regulated antioxidative signaling pathway in luteolin-induced prevention of the diabetic rat heart from ischemia/reperfusion injury.

Luteolin attenuates myocardial ischemia/reperfusion (I/R) injury in diabetes through activating the nuclear factor erythroid 2-related factor 2 (Nrf2)-related antioxidative response. Though sestrin2, a highly conserved stress-inducible protein, is regarded as a modulator of Nrf2 and reduces I/R injury, the effect of sestrin2 on luteolin-induced prevention of the diabetic heart from I/R injury remains unclear. We hypothesized that luteolin could relieve myocardial I/R injury in diabetes by activating the sestrin2-modulated Nrf2 antioxidative response. Diabetes was induced in rats using a single dose of streptozotocin (65 mg kg-1, i.p.) for 6 weeks, and then luteolin (100 mg kg-1 d-1, i.g.), Nrf2 inhibitor brusatol, or sestrin2 blocker leucine was administered for 2 consecutive weeks. After that, the hearts were isolated and exposed to global I/R (30 min/120 min). Luteolin markedly improved cardiac function, myocardial viability and expressions of Nrf2-regulated antioxidative genes, and reduced lactate dehydrogenase release, malondialdehyde, and 8-hydroxydeoxyguanosine in the diabetic I/R hearts. Ca2+-induced mitochondrial permeability transition and membrane potential disruption were markedly inhibited in luteolin-treated diabetic ventricular myocytes. All these effects of luteolin were significantly reversed by Nrf2 inhibitor brusatol or sestrin2 inhibitor leucine. Luteolin-induced diminished Keap1 and augmented nuclear translocation and ARE binding activity of Nrf2 were hampered by leucine in the diabetic I/R heart. In addition, luteolin-induced augmented transcription of sestrin2 was markedly blocked by brusatol in the diabetic I/R heart. These data suggest that sestrin2 and Nrf2 positively interact to promote antioxidative actions and attenuate mitochondrial damage, by which luteolin relieves diabetic myocardial I/R injury.

Nanoleakage and microtensile bond strength at the adhesive-dentin interface after different etching times.

PURPOSE: To evaluate the effect of etching time on the nanoleakage and microtensile bond strength (microTBS) at the interface between etch-and-rise adhesives and dentin. METHODS: Eighty molars were sectioned to expose the superficial dentin and randomly divided into four groups according to the adhesives used: OptiBond Solo (OB), Single Bond (SB), One-Step (OS), and Prime&Bond NT (PB). Within each group, a total-etching technique was applied, and the dentin surfaces were etched for 0, 15, 30 or 60 seconds. Each treated tooth was then sectioned into sticks. A non-trimming microTBS test and TEM observation for nanoleakage were sequentially performed. RESULTS: The degree of nanoleakage increased as etching time increased (P < 0.05). For the OS and PB groups, the highest microTBS values were achieved with 15 seconds of etching, followed by 30, 60 and 0 seconds. For the OB and SB groups, the microTBS values for 15- and 30-second etching times were similar and were both significantly higher than those of 0 and 60 seconds (P < 0.05).

Luteolin Attenuates Cardiac Ischemia/Reperfusion Injury in Diabetic Rats by Modulating Nrf2 Antioxidative Function.

Luteolin has been reported to attenuate ischemia/reperfusion (I/R) injury in the diabetic heart through endothelial nitric oxide synthase- (eNOS-) related antioxidative response. Though the nuclear factor erythroid 2-related factor 2 (Nrf2) is regarded as a key endogenous factor to reduce diabetic oxidative stress, whether luteolin reduces cardiac I/R injury in the diabetic heart via enhancing Nrf2 function needs to be clarified. We hypothesized that pretreatment with luteolin could alleviate cardiac I/R injury in the diabetic heart by affecting the eNOS/Nrf2 signaling pathway. The diabetic rat was produced by a single injection of streptozotocin (65 mg/kg, i.p.) for 6 weeks, and then, luteolin (100 mg/kg/day, i.g.), eNOS inhibitor L-NAME, or Nrf2 inhibitor brusatol was administered for the succedent 2 weeks. After that, the isolated rat heart was exposed to 30 min of global ischemia and 120 min of reperfusion to establish I/R injury. Luteolin markedly ameliorated cardiac function and myocardial viability; upregulated expressions of heme oxygenase-1, superoxide dismutase, glutathione peroxidase, and catalase; and reduced myocardial lactate dehydrogenase release, malondialdehyde, and 8-hydroxydeoxyguanosine in the diabetic I/R heart. All these ameliorating effects of luteolin were significantly reversed by L-NAME or brusatol. Luteolin also markedly reduced S-nitrosylation of Kelch-like ECH-associated protein 1 (Keap1) and upregulated Nrf2 and its transcriptional activity. This effect of luteolin on Keap1/Nrf2 signaling was attenuated by L-NAME. These data reveal that luteolin protects the diabetic heart against I/R injury by enhancing eNOS-mediated S-nitrosylation of Keap1, with subsequent upregulation of Nrf2 and the Nrf2-related antioxidative signaling pathway.

Luteolin alleviates cardiac ischemia/reperfusion injury in the hypercholesterolemic rat via activating Akt/Nrf2 signaling.

Myocardial ischemia/reperfusion (I/R) injury in hypercholesterolemia is associated with oxidative stress, while luteolin is known to reduce oxidative stress by activating Akt/nuclear factor erythroid-2-related factor 2 (Nrf2) signaling and alleviate cardiac I/R injury. Here, we investigated whether luteolin pretreatment diminishes myocardial I/R injury in hypercholesterolemic rats by activating Akt/Nrf2 signaling. Hypercholesterolemic rats were produced by 2% cholesterol diet for 8 weeks. Luteolin (100 mg/kg/day, i.g.) or LY294002 was administered for the last 2 weeks. The hearts were then isolated and subjected to 30 min of global ischemia followed by 120 min of reperfusion. Pretreatment with luteolin significantly improved left ventricular function throughout reperfusion, increased cardiac tissue viability, reduced coronary lactate dehydrogenase release and the myocardial malondialdehyde level, upregulated p-Akt and p-GSK3ß expressions, inhibited nuclear translocation of Fyn, and activated Nrf2 function in hypercholesterolemic I/R rat hearts. All these improving effects of luteolin were significantly attenuated by LY294002. Ca2+-induced mitochondrial permeability transition pore (mPTP) opening and mitochondrial inner membrane potential reduction were significantly inhibited in ventricular myocytes isolated from luteolin-treated hypercholesterolemic rats, which were attenuated by LY294002. These results indicate that luteolin protects the hypercholesterolemic heart against I/R injury due to upregulation of Akt-mediated Nrf2 antioxidative function and inhibition of mPTP.

Chlamydial infection among patients attending STD and genitourinary clinics in Taiwan.

BACKGROUND: The main objective of this study is to examine the epidemiology of Chlamydia trachomatis (CT) infection amongst patients (473 men, 180 women) seen two hospitals in Taiwan. METHODS: Between July 2004 and June 2005, a total of 653 patients provided first-void urine samples for examination of CT using PCR assay. RESULTS: The overall prevalence of CT infection was 18.4% (95% confidence interval [CI] 17.3-19.5). Prevalence for men and women were 16.7 % (95% CI 15.3-18.0%) and 22.8% (95% CI 17.5-28.1%), respectively. Age group-specific prevalence was 25.7% (95% CI 22.5-28.9%) in < 20 year olds, 23.5% (95% CI 20.3-26.7%) in 20-24 year olds, 22.3% (95% CI 18.9-25.7%) in 25-30 year olds, and 11.5% (95% CI 10.3-12.7%) in > 30 year olds. Independent risk factors for chlamydial infection included younger age (aged < or = 30 years) (adjusted odds ratio [AOR] = 2.44; 95% CI 1.52-3.84; p < 0.001), inconsistent condom use (AOR = 2.01; 95% CI 1.32-3.06; p < 0.001), being symptomatic (dysuria, urethral discharge) at the time of testing (AOR = 1.84; 95% CI 1.21-2.80; p < 0.001), and having N. gonorrhoeae infection (AOR = 3.82; 95% CI 2.20-6.58; p < 0.001). CONCLUSION: Genital chlamydial infection is an important sexually transmitted disease in Taiwan. Young Taiwanese persons attending a STD clinic should be screened for CT infection and counselled on condom use.

Effects of CYP3A4 polymorphisms on efficiency of general anesthesia combined with epidural block in patients undergoing cardiac valve replacement.

This study aims to investigate the effects of CYP3A4 polymorphisms (*4, *5 and *6) on efficiency of general anesthesia (GA) combined with epidural block (EB) in patients undergoing cardiac valve replacement. From January 2014 to October 2015, a total of 511 patients undergoing cardiac valve replacement (case group) and 503 healthy individuals (control group) were selected for the study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied for genotyping of CYP3A4 gene. Central venous pressure (CVP), mean arterial pressure (MAP), heart rate (HR), pulse oximetry (SPO2), extubation and duration of intensive care unit (ICU) stay during the surgery were observed and recorded. A nine-month follow-up was conducted. Genotype and allele frequency of CYP3A4*4 were significantly different between the case and control groups (all P < 0.05). Compared with wild-type *1*1 patients with heterozygous *1*4 of CYP3A4*4 showed significant difference in HR, MAP, SPO2 and CVP and in the time of extubation and ICU stay. CYP3A4*4 polymorphism may be associated with the effect of GA combined with EB in cardiac surgery. These results demonstrate that CYP3A4*4 polymorphism is correlated with the effects of GA combined with EB in cardiac surgery. CYP3A4 polymorphisms increase the risk of GA combined with EB among patients undergoing cardiac valve replacement.

Effect of X-ray irradiation on hepatocarcinoma cells and erythrocytes in salvaged blood.

The broad clinical acceptance of intraoperative blood salvage and its applications in cancer surgery remain controversial. Until now, a method that can safely eliminate cancer cells while preserving erythrocytes does not exist. Here, we investigated whether X-ray generated from linear accelerator irradiation at a certain dose can kill hepatocarcinoma cells while preserving erythrocytes. HepG2, SK-Hep1 or Huh7 cells were mixed into the aliquots of erythrocytes obtained from healthy volunteers. After the mixed cells were exposed to 30 Gy and 50 Gy X-rays irradiation, the viability, clonogenicity, DNA synthesis and tumorigenicity of the tumor cells were determined by the MTT assay, plate colony formation, 5-ethynyl-2'-deoxyuridine incorporation, and subcutaneous xenograft implantation into immunocompromised mice. The ATP, 2,3-DPG, free Hb, osmotic fragility, blood gas variables in erythrocytes and morphology of erythrocytes at 0 h, 12 h, 24 h, 48 h, 72 h after irradiation were analyzed. X-ray irradiation at 30 Gy effectively inhibited the viability, proliferation, and tumorigenicity of HepG2, SK-Hep1 and Huh7 cells without noticeably damaging the ability of oxygen-carrying, membrane integrity and morphology of erythrocytes. Theses results suggest that X-ray at 30 Gy irradiation might be safe to eliminate hepatocarcinoma cells while preserving erythrocytes in salvaged blood.

Irradiation Can Selectively Kill Tumor Cells while Preserving Erythrocyte Viability in a Co-Culture System.

An understanding of how to safely apply intraoperative blood salvage (IBS) in cancer surgery has not yet been obtained. Here, we investigated the optimal dose of 137Cs gamma-ray irradiation for killing human hepatocarcinoma (HepG2), gastrocarcinoma (SGC7901), and colonic carcinoma (SW620) tumor cells while preserving co-cultured erythrocytes obtained from 14 healthy adult volunteers. HepG2, SGC7901, or SW620 cells were mixed into the aliquots of erythrocytes. After the mixed cells were treated with 137Cs gamma-ray irradiation (30, 50, and 100 Gy), tumor cells and erythrocytes were separated by density gradient centrifugation in Percoll with a density of 1.063 g/ml. The viability, clonogenicity, DNA synthesis, tumorigenicity, and apoptosis of the tumor cells were determined by MTT assay, plate colony formation, 5-ethynyl-2'-deoxyuridine (EdU) incorporation, subcutaneous xenograft implantation into immunocompromised mice, and annexin V/7-AAD staining, respectively. The ATP concentration, 2,3-DPG level, free Hb concentration, osmotic fragility, membrane phosphatidylserine externalization, blood gas variables, reactive oxygen species levels, and superoxide dismutase levels in erythrocytes were analyzed. We found that 137Cs gamma-ray irradiation at 50 Gy effectively inhibited the viability, proliferation, and tumorigenicity of HepG2, SGC7901, and SW620 cells without markedly damaging the oxygen-carrying ability or membrane integrity or increasing the oxidative stress of erythrocytes in vitro. These results demonstrated that 50 Gy irradiation in a standard 137Cs blood irradiator might be a safe and effective method of inactivating HepG2, SGC7901, and SW620 cells mixed with erythrocytes, which might help to safely allow IBS in cancer surgery.

Cisplatin combined with hyperthermia kills HepG2 cells in intraoperative blood salvage but preserves the function of erythrocytes.

The safe use of intraoperative blood salvage (IBS) in cancer surgery remains controversial. Here, we investigated the killing effect of cisplatin combined with hyperthermia on human hepatocarcinoma (HepG2) cells and erythrocytes from IBS in vitro. HepG2 cells were mixed with concentrated erythrocytes and pretreated with cisplatin (50, 100, and 200 µg/ml) alone at 37 °C for 60 min and cisplatin (25, 50, 100, and 200 µg/ml) combined with hyperthermia at 42 °C for 60 min. After pretreatment, the cell viability, colony formation and DNA metabolism in HepG2 and the Na(+)-K(+)-ATPase activity, 2,3-diphosphoglycerate (2,3-DPG) concentration, free hemoglobin (Hb) level, osmotic fragility, membrane phosphatidylserine externalization, and blood gas variables in erythrocytes were determined. Pretreatment with cisplatin (50, 100, and 200 µg/ml) combined with hyperthermia (42 °C) for 60 min significantly decreased HepG2 cell viability, and completely inhibited colony formation and DNA metabolism when the HepG2 cell concentration was 5×10(4) ml(-1) in the erythrocyte (P<0.01). Erythrocytic Na(+)-K(+)-ATPase activity, 2,3-DPG level, phosphatidylserine externalization, and extra-erythrocytic free Hb were significantly altered by hyperthermia plus high concentrations of cisplatin (100 and 200 µg/ml) (P<0.05), but not by hyperthermia plus 50 µg/ml cisplatin (P>0.05). In conclusion, pretreatment with cisplatin (50 µg/ml) combined with hyperthermia (42 °C) for 60 min effectively eliminated HepG2 cells from IBS but did not significantly affect erythrocytes in vitro.

Molecular epidemiology of genital chlamydial infection among male patients attending an STD clinic in Taipei, Taiwan.

OBJECTIVE: To assess the epidemiology of genital Chlamydia trachomatis infection among men attending an STD clinic in Taipei, Taiwan. STUDY: Between July 2004 and June 2005, a total of 426 first-void urine specimens from male patients were tested for C. trachomatis by using a urine-based PCR DNA amplification assay. RESULTS: The overall prevalence of genital C. trachomatis infection was 16.4%. Youth, current symptoms, the presence of N. gonorrhoeae infection, and inconsistent use of condoms were positively associated with C. trachomatis infection. The most common chlamydia strain present was genotype E, followed by D and Da, F, K, J, G, and H. CONCLUSIONS: C. trachomatis genital infection was prevalent among male patients attending a STD clinic in Taipei. Young Taiwanese men attending STD clinics should be counselled on condom use.

[The influence of nanometer inorganic filling carrying silver on the properties of composite resin].

PURPOSE: To observe the mechanical performance's effects of nanometer inorganic filling carrying silver on the properties of composite resin and find the perfect dose of the mixture. METHODS: The resin was mixed with inorganic filling whose doses were 0.5%, 1.0%, 1.5% and 2.0%, and then the compressive strength, flexure strength and wear-resistance were measured in the five groups. One-way ANOVA and Student's t test were used for statistical analysis in SPSS 10.0. RESULTS: The results showed that in the compressive strength tests, the 1.5% dose was perfect which was (264.93+/-12.48) MPa (P>0.05) and the 2.0% dose was bad which was (94.54+/-17.42) MPa (P<0.01); in the flexure strength tests, 0.5%, 1.0% and 1.5% dose didn't change significantly which were (86.25+/-12.76) MPa, (99.49+/-16.47) MPa and (108.69+/-10.35) MPa (P>0.05) respectively, and 2.0% dose went down significantly which was (79.79+/-8.10) MPa (P<0.01); in the wear-resistance tests, 1.5% and 2.0% worked very well which were (2.73+/-0.53) MPa (P<0.05), (2.70+/-1.25) MPa (P<0.01) respectively. CONCLUSION: Nanometer inorganic filling had no significant influence on the composite resin, and 1.5% dose of nanometer inorganic filling is perfect for the resin's mechanical properties.

[The study on fabrication of dental restoration using PMMA-ZrO2 composites via CAD/CAM].

OBJECTIVE: To obtain dental restorations by machining PMMA-ZrO2 organic-inorganic composites with the dental CAD/CAM system. METHODS: Partially sintered Zirconia compacts (PSZC) were prepared via isostatic pressing and partially sintering, with Zirconia nanopowder as raw materials. PMMA-Zirconia organic-inorganic composites were prepared by vacuum infiltrating the prepolymerized MMA into the PSZC, followed by in-situ polymerization. The mechanical properties and machinability of composites were studied. The composites were machined on the dental CAD/CAM system to obtain dental restoration. RESULTS: At 71.44% TD of PSZC, the composite had a 3-point bending strength of (202.56 +/- 3.09) MPa, fracture toughness of (4.30 +/- 0.16) MPa.m(1/2), elasticity modulus of (58.71 +/- 1.98) GPa, and Vickers hardness of (3.82 +/- 0.34) GPa, respectively. A premolar crown was fabricated by CAD/CAM system in 16 mins, and was verisimilitude, without any cracks. CONCLUSIONS: The composite at 71.44% TD of PSZC has good mechanical properties and dental restorations can be manufactured by PMMA-Zirconia composites via dental CAD/CAM system.

[A study of injectable autogenous tissue-engineered bone].

OBJECTIVE: To investigate the utilization of carrier for delivering osteoblasts and creating autogenous bone tissue in ectopic site of animal via injection. METHODS: Bone marrow cells harvested from iliac bone of New Zealand rabbits were induced to differentiate into marrow stromal osteoblasts. The osteoblasts were mixed with 1.5% alginate sodium solution to generate osteoblasts/alginate composites with final cellular density of 4 x 10(9)/L. Calcium chloride was used as cross-linking agent to gel aqueous alginate solution. The marrow stromal osteoblasts/alginate composites were injected into the dorsal subcutaneous tissue of rabbits with autogenous cells transplantation. The samples were examined with X-ray and histological analysis. RESULTS: Four, eight and twelve weeks after injection, the hard knobbles were easily palpated under the dorsal skin of animals. On X-ray photograph the samples showed calcified image with more density than surrounding soft tissue, new bone formation was observed in the osteoblasts/alginate composites in histological analysis. The osteogenesis was in association with regenerated hematopoietic bone marrow. CONCLUSIONS: These results demonstrate that new bone tissue could be created through the injection of alginate sodium treated with autogenous marrow stromal osteoblasts.