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Purpose To investigate the effect of prolonged sevoflurane (SEV) exposure on differentiation potential and hypoxia tolerance of neural stem cells (NSCs). Materials and methods NSCs were extracted from 15-day fetal mice. After sub-culture, Sevoflurane exposure treatment was performed. Cell cycle were detected by flow cytometry. Western Blot and immunofluorescence assay were used to detect the expression and spatial distribution of Nestin, NSE, GFAP, Oct4, and SOX2; CCK-8 detected cell viability. Cell growth morphology was observed under a microscope. Tunel detected cell apoptosis; the concentration of extracel-lular lactate dehydrogenase (LDH) was determined by ELISA. Results Compared with the control group, the proportion of NSCs in the G2/M phase increased in the Sevoflurane exposure group; our results also suggested the sphere- formation rate decreased significantly, Increased apoptosis and decreased cell viability; Besides, the level of LDH release increased. Conclusion Long-term exposure to sevoflurane (> 8 h) promoted the premature differentiation of NSCs and reduced their pluripotency, reserves, and hypoxia tolerance. This study reveals the reasons underlying damage to the nervous system of young children induced by long-term exposure to sevoflurane from the perspective of CNS reserve cells.

The purpose of this study was to determine whether the epidermal growth factor receptor (EGFR), which is a classical receptor tyrosine kinase, is involved in the protective effect of morphine against ischemia/reperfusion (I/R)-induced myocardial mitochondrial damage. Isolated rats hearts were subjected to global ischemia followed by reperfusion. Cardiac H9c2 cells were exposed to a simulated ischemia solution followed by Tyrode’s solution to induce hypoxia/reoxygenation (H/R) injury. Triphenyltetrazolium chloride (TTC) was used to measure infarct size. The mitochondrial morphological and functional changes were determined using transmission election microscopy (TEM), mitochondrial stress assay, and mitochondrial swelling, respectively. Mitochondrial fluorescence indicator JC-1, DCFH-DA, and Mitosox Red were used to determine mitochondrial membrane potential (△Ψm), intracellular reactive oxygen species (ROS) and mitochondrial superoxide. A TUNUL assay kit was used to detect the level of apoptosis. Western blotting analysis was used to measure the expression of proteins. Treatment of isolated rat hearts with morphine prevented I/R-induced myocardial mitochondrial injury, which was inhibited by the selective EGFR inhibitor AG1478, suggesting that EGFR is involved in the mitochondrial protective effect of morphine under I/R conditions. In support of this hypothesis, the selective EGFR agonist epidermal growth factor (EGF) reduced mitochondrial morphological and functional damage similarly to morphine. Further study demonstrated that morphine may alleviate I/R-induced cardiac damage by inhibiting autophagy but not apoptosis. Morphine increased protein kinase B (Akt), extracellular regulated protein kinases (ERK) and signal transducer and activator of transcription-3 (STAT-3) phosphorylation, which was inhibited by AG1478, and EGF had similar effects, indicating that morphine may activate Akt, ERK, and STAT-3 via EGFR. Morphine and EGF increased intracellular reactive oxygen species (ROS) generation. This effect of morphine was inhibited by AG1478, indicating that morphine promotes intracellular ROS generation by activating EGFR. However, morphine did not increase ROS generation when cells were transfected with siRNA against EGFR. In addition, EGFR activity was markedly increased by morphine, but the effect of morphine was reversed by naltrindole. These results suggest that morphine may activate EGFR via δ-opioid receptor activation. Morphine may prevent I/R-induced myocardial mitochondrial damage by activating EGFR through δ-opioid receptors, in turn increasing RISK and SAFE pathway activity via intracellular ROS. Moreover, morphine may reduce myocardial injury by regulating autophagy but not apoptosis.

We evaluated the ability of different fluorescent indicators by various analytical instruments, including a laser scanning confocal microscope (LSCM), fluorescence plate reader, and flow cytometer (FCM), to measure the mitochondrial membrane potential (ΔΨm) of cardiac H9c2 cells during oxidative stress-induced mitochondrial injury. The mitochondrial oxygen consumption rate and a transmission electron microscope were used to detect changes in mitochondrial functions and morphology, respectively. Cardiac H9c2 cells were exposed to H2O2 (500, 750, 1000, and 1250 μM) to induce mitochondrial oxidative stress injury, and fluorescent indicators including tetramethyl rhodamine ethyl ester (TMRE), 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolocarbocyanine iodide (JC-1), and rhodamine 123 (R123) were used to detect changes in ΔΨm using an LSCM, fluorescence plate reader, and FCM. The decrease in ΔΨm caused by H2O2 was determined by endpoint and dynamic analyses after staining with JC-1 or TMRE. With the R123 probe, the LSCM could only detect the change in ΔΨm caused by 1000 μM H2O2. Moreover, R123 was less effective than JC-1 and TMRE for measurement of ΔΨm by the LSCM. Our data indicated that an LSCM is the most suitable instrument to detect dynamic changes in ΔΨm, whereas all three instruments can detect ΔΨm at the endpoint.

Primary hepatocellular carcinoma (PHC) is a major health problem worldwide and is one of the 10 most commonly diagnosed cancers in China. Heat shock protein 27 (HSP27) were found to be overexpressed in a wide range of malignancies including PHC, however, post-translational modification of HSP27 still needs exploration in PHC. Recently, SUMOylation, an important post-translational modification associating with the development of many kinds of cancers has been intensively studied. In the current study, mRNA and protein level of HSP27 in archived tumor samples representing various pathological characteristics of PHC were examined, and modification of HSP27 by SUMO2/3 was investigated. HSP27 were expressed abundantly in patients' tumor tissues, and found to be associated with pathological progression. Besides, HSP27 was also elevated significantly in liver cancer cell lines Huh7 and HepG2 compared with human hepatocyte cells L02. Furthermore, knockdown of HSP27 was found to be associated with the decreased proliferation and invasion ability in Huh7 and HepG2 cells. Immunofluorescence assay showed that HSP27 and SUMO2/3 were co-localized in the subcellular, and co-immunoprecipitation verified the interaction between HSP27 and SUMO2/3. Overexpression of SUMO2/3 upregulated the HSP27 protein level and promotes Huh7 and HepG2 cell proliferation and invasion, and vice versa when the SUMO2/3 was knockdown. Taken together, increased protein level of HSP27 through SUMO2/3-mediated SUMOylation plays crucial roles in the progression of PHC, and this finding may shed light on developing potential therapeutic targets for PHC.

While it is well known that adenosine receptor activation protects the heart from ischemia/reperfusion injury, the precise mitochondrial mechanism responsible for the action remains unknown. This study probed the mitochondrial events associated with the cardioprotective effect of 5'-(N-ethylcarboxamido) adenosine (NECA), an adenosine A2 receptor agonist. Isolated rat hearts were subjected to 30min ischemia followed by 10min of reperfusion, whereas H9c2 cells experienced 20min ischemia and 10min reperfusion. NECA prevented mitochondrial structural damage, decreases in respiratory control ratio (RCR), and collapse of mitochondrial membrane potential (ΔΨm). Both the adenosine A2A receptor antagonist SCH58261 and A2B receptor antagonist MRS1706 inhibited the action of NECA. NECA reduced mitochondrial proteins carbonylation, H2O2, and superoxide generation at reperfusion, but did not change superoxide dismutase (SOD) activity. In support, the protective effects of NECA and Peg-SOD on ΔΨm upon reperfusion were additive, implying that NECA's protection is attributable to the reduced superoxide generation but not to the enhancement of the superoxide-scavenging capacity. NECA increased the mitochondrial Src tyrosine kinase activity and suppressed complex I activity at reperfusion in a Src-dependent manner. NECA also reduced mitochondrial superoxide through Src tyrosine kinase. Studies with liquid chromatography-mass spectrometer (LC-MS) identified Tyr118 of the NDUFV2 subunit of complex 1 as a likely site of the tyrosine phosphorylation. Furthermore, the complex I activity of cells transfected with the Y118F mutant was increased, suggesting that this site might be a negative regulator of complex I activity. In support, NECA failed to suppress complex I activity at reperfusion in cells transfected with the Y118F mutant of NDUFV2. In conclusion, NECA prevents mitochondrial oxidative stress by decreasing mitochondrial superoxide generation through inhibition of complex I via the mitochondrial Src tyrosine kinase. Phosphorylation of Tyr118 residue in NDUFV2 subunit may account for the inhibitory effect of NECA on complex I.

Background. Zinc plays a role in mitophagy and protects cardiomyocytes from ischemia/reperfusion injury. This study is aimed at investigating whether SUMOylation of Drp1 is involved in the protection of zinc ion on cardiac I/R injury. Methods. Mouse hearts were subjected to 30 minutes of regional ischemia followed by 2 hours of reperfusion (ischemia/reoxygenation (I/R)). Infarct size and apoptosis were assessed. HL-1 cells were subjected to 24 hours of hypoxia and 6 hours of reoxygenation (hypoxia/reoxygenation (H/R)). Zinc was given 5 min before reperfusion for 30 min. SENP2 overexpression plasmid (Flag-SENP2), Drp1 mutation plasmid (Myc-Drp1 4KR), and SUMO1 siRNA were transfected into HL-1 cells for 48 h before hypoxia. Effects of zinc on SUMO family members were analyzed by Western blotting. SUMOylation of Drp1, apoptosis and the collapse of mitochondrial membrane potential (ΔΨm), and mitophagy were evaluated. Results. Compared with the control, SUMO1 modification level of proteins in the H/R decreased, while this effect was reversed by zinc. In the setting of H/R, zinc attenuated myocardial apoptosis, which was reversed by SUMO1 siRNA. Similar effects were observed in SUMO1 KO mice exposed to H/R. In addition, the dynamin-related protein 1 (Drp1) is a target protein of SUMO1. The SUMOylation of Drp1 induced by zinc regulated mitophagy and contributed to the protective effect of zinc on H/R injury. Conclusions. SUMOylation of Drp1 played an essential role in zinc-induced cardio protection against I/R injury. Our findings provide a promising therapeutic approach for acute myocardial I/R injury.

Common genetic variants (single-nucleotide polymorphisms [SNPs]) in microRNA genes may alter their maturation or expression, resulting in varied functional consequences. Several studies have evaluated the association between the SNP rs11614913 and cancer risk in diverse populations and in a range of cancers, with contradictory outcomes. In this study, we examined 114 paired samples (tumor and normal tissues) from breast cancer patients to study the genotype distribution and somatic mutation of the SNP in MIR 196A2 (rs11614913 C-T). In addition, we evaluated their influence on the mature MIR 196A2 expression. We found that 14% (16/114) of tumors underwent somatic mutation of the SNP rs11614913. Moreover, the CT heterozygous and the CC homozygous states of SNP rs11614913 were more prone to mutation, while the TT homozygous state appeared to be resistant. We further detected a significant increase (p = 0.002) in mature MIR 196A2 expression in breast cancer. In particular, we found a significant association between the occurrence of SNP rs11614913 mutation and high expression (p = 0.0002). In addition, the mature MIR 196A2 expression level was significantly associated with the higher tumor grade (p = 0.004). Taken together, our results seem to demonstrate that somatic mutation of SNP rs11614913 in MIR 196A2 can have an influence on its expression. In addition, it indicated that an unknown mechanism is responsible for both the mutation of SNP rs11614913 and the dysregulation of mature MIR 196A2 expression.

OBJECTIVE: To explore the role of mitochondrial permeability transition pore (mPTP) in mediating the protective effect of gastrodin against oxidative stress damage in H9c2 cardiac myocytes. METHODS: H9c2 cardiac myocytes were treated with H(2)O(2), gastrodin, gastrodin+H(2)O(2), cyclosporin A (CsA), or CsA+gas+H(2)O(2) group. MTT assay was used to detect the survival ratio of H9c2 cells, and flow cytometry with Annexin V-FITC/PI double staining was used to analyze the early apoptosis rate after the treatments. The concentration of ATP and level of reactive oxygen species (ROS) in the cells were detected using commercial kits. The mitochondrial membrane potential of the cells was detected with laser confocal microscopy. The expression of cytochrome C was detected with Western blotting, and the activity of caspase-3 was also assessed in the cells. RESULTS: Gastrodin pretreatment could prevent oxidative stress-induced reduction of mitochondrial membrane potential, and this effect was inhibited by the application of CsA. Gastrodin significantly lowered the levels of ROS and apoptosis-related factors in H(2)O(2)-exposed cells, and such effects were reversed by CsA. CsA significantly antagonized the protective effect of gastrodin against apoptosis in H(2)O(2)-exposed cells. CONCLUSIONS: Gastrodin prevents oxidative stress-induced injury in H9c2 cells by inhibiting mPTP opening to reduce the cell apoptosis.

OBJECTIVE: Although several methods have been used to detect the intracellular reactive oxygen species (ROS) generation, it is still difficult to determine where ROS generate from. This study aimed to demonstrate whether ROS generate from mitochondria during oxidative stress induced mitochondria damage in cardiac H9c2 cells by laser scanning confocal microscopy (LSCM). METHODS: Cardiac H9c2 cells were exposed to H2 O2 (1200μM) to induce mitochondrial oxidant damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE); ROS generation was measured by staining cells with dichlorodihydrofluorescein diacetate (H2 DCFDA). RESULTS: A rapid/transient ROS burst from mitochondria was induced in cardiac cells treated with H2 O2 compared with the control group, suggesting that mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells. Meanwhile, the TMRE fluorescence intensity of mitochondria which had produced a great deal of ROS decreased significantly, indicating that the burst of ROS induces the loss of ΔΨm. In addition, the structure of mitochondria was damaged seriously after ROS burst. However, we also demonstrated that the TMRE fluorescence intensity might be affected by H2 DCFDA. CONCLUSIONS: Mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells and these findings provide a new method to observe whether ROS generate from mitochondria by LSCM. However, these observations also suggested that it is inaccurate to test the fluorescence intensities of cells stained with two or more different fluorescent dyes which should be paid more attention to.

Although Akt is reported to play a role in morphine's cardioprotection, little is known about the mechanism underlying morphine-induced Akt activation. This study aimed to define the molecular mechanism underlying morphine-induced Akt activation and to determine if the mechanism contributes to the protective effect of morphine on ischemia/reperfusion injury. In cardiac H9c2 cells, morphine increased Akt phosphorylation at Ser(473), indicating that morphine upregulates Akt activity. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a major regulator of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling, was not involved in the action of morphine on Akt activity. Morphine decreased the activity of PP2A, a major protein Ser/Thr phosphatase, and inhibition of PP2A with okadaic acid (OA) mimicked the effect of morphine on Akt activity. The effects of morphine on PP2A and Akt activities were inhibited by the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)glycine (MPG) and the mitochondrial K(ATP) channel closer 5-hydroxydecanoate (5HD). In support, morphine could produce ROS and this was reversed by 5HD. Finally, the cardioprotective effect of morphine on ischemia-reperfusion injury was mimicked by OA but was suppressed by 5HD or MPG, indicating that protein phosphatases and ROS are involved in morphine's protection. In conclusion, morphine upregulates Akt activity by inactivating protein Ser/Thr phosphatases via ROS, which may contribute to the cardioprotective effect of morphine.

Although several methods have been used to detect the intracellular reactive oxygen species (ROS) generation, it is still difficult to determine where ROS generate from. This study aimed to demonstrate whether ROS generate from mitochondria during oxidative stress induced mitochondria damage in cardiac H9c2 cells by laser scanning confocal microscopy (LSCM).Cardiac H9c2 cells were exposed to H2 O2 (1200μM) to induce mitochondrial oxidant damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE); ROS generation was measured by staining cells with dichlorodihydrofluorescein diacetate (H2 DCFDA).A rapid/transient ROS burst from mitochondria was induced in cardiac cells treated with H2 O2 compared with the control group, suggesting that mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells. Meanwhile, the TMRE fluorescence intensity of mitochondria which had produced a great deal of ROS decreased significantly, indicating that the burst of ROS induces the loss of ΔΨm. In addition, the structure of mitochondria was damaged seriously after ROS burst. However, we also demonstrated that the TMRE fluorescence intensity might be affected by H2 DCFDA.Mitochondria are the main source of ROS induced by oxidative stress in H9c2 cells and these findings provide a new method to observe whether ROS generate from mitochondria by LSCM. However, these observations also suggested that it is inaccurate to test the fluorescence intensities of cells stained with two or more different fluorescent dyes which should be paid more attention to.

Effects of technological innovation on performance are significantly important to firm's competitive advantages. This paper aims to find the key points when studying effects of technological innovation on performance, and to build a conceptual model. The results show that R&D and marketing capabilities are critical. In the conceptual model, not only effects of R&D and marketing capabilities, but also integration of two capabilities should be included. The paper will contribute to the further empirical research in this field.

Objectives Glycogen synthesis kinase-3β (GSK-3 β ) has been widely proved to be linked to the inhibition of the mPTP opening. This study aimed to determine whether puerarin prevents oxidative stress-induced mitochondrial damage via inhibiting GSK-3β activity. Methods Cardiac H9c2 cells were exposed to H 2 O 2 for 20 min to induce mitochondrial oxidative damage. Mitochondrial membrane potential (ΔΨm) was measured by staining cells with tetramethylrhodamine ethyl ester (TMRE). V-akt-murinet hymoma viral onco-gene homolog (Akt), and GSK-3 β phosphorylation were measured with Western blotting. Results Treatment of cardiac cells with puerarin prevented H 2 O 2 -induced loss of ΔΨm suggesting that puerarin prevents oxidative stress-induced mitochondrial damage. Puerarin did not alter the Aktphosphorylation, indicating that Aktmay not be involved in the cardioprotective effect of puerarin. However, treatment of cardiac cells with puerarin significantly increased GSK-3β phosphorylation, indicating that puerarin may prevent oxidative stress-induced myocardial injury through downregulating GSK-3β activity. Conclusions Puerarin prevents oxidative stress-induced mitochondrial damage via inhibiting GSK-3β activity.

Objectives This study aimed to test the protection of puerarin in oxidative stress-induced mitochondrial injury in rat cardiac H9c2 cells. Methods Rat heart tissue-derived H9c2 cells were used. First, we test if puerarin could modulate the mPTP opening. To detect the mPTP opening, cells were loaded with TMRE and imaged with confocal microscopy. After treatments with puerarin at different concentrations, cells were exposed to 650 µM H 2 O 2 to induce the mPTP opening, and were tested by the MTT assay to detect cell activity. To define the signalling mechanism responsible for the protective effect of puerarin, phosphor-ylation status of SAPK/JNK and p38MAPK were determined with Western blot. Results Cardiac H9c2 cells treated with 0.1 µM puerarin showed a significant increase in TMRE fluorescence intensity compared to the control group, indicating that puerarin can prevent oxidant-induced mitochondrial damage. MTT assay were used to detect cell activity. Compared to the control group, cells treated with puerarin (0.1 µM) showed a significantly increased in absorbance ratio, indicating the cardioprotective effect of puerarin. The effect of H 2 O 2 on TMRE fluorescence was inhibited by SP600125, implying that the JNK signalling pathways may mediate the action of Puerarin. Compared to the control group, cells treated with puerarin (0.1 µM) showed a significant decrease in phosphorylation of SAPK/JNK and did not alter the phosphorylated p38MAPK, indicating that JNK mediates the cardioprotective effect of puerarin. Conclusions Puerarin may restrain oxidative stress-induced injury through inhibit ing JNK phosphorylation.

Objectives Aims We previously documented that morphine upregulates Akt activity by inactivating protein Ser/Thr phosphatases via reactive oxygen species (ROS) may contribute to the protective effect of morphine on ischaemia/reperfusion injury, but the molecular mechanism underlying morphine-induced ROS production remains to be defined. Since receptor tyrosine kinases (RTKs) may contribute to the cardiac protection induced by the activation of opioid, bradykinin, or muscarinic receptors and could increase intracellular ROS production, the purpose of this study was to determine whether RTKs are involved in the protective effect of morphine against oxidant stress-induced mitochondrial damage and whether morphine could induce intracellular ROS production via RTKs, focusing on the roles of insulin-like growth factor 1-receptor tyrosine kinase (IGF-1RTK) and epidermal growth factor receptor tyrosine kinase (EGFRTK). Methods Cardiac H9c2 cells were exposed to H 2 O 2 for 20 min to induce mitochondrial oxidant damage and confocal microscopy was used to determine whether morphine could prevent the oxidant stress-induced loss of mitochondrial membrane potential (ΔΨm) by monitoring changes in TMRE fluorescence. ROS generation was measured by H 2 DCFDA kit. Western blot and immunofluorescence assay were used to test Akt and EGFRTK phosphorylation. Results Treatment of cardiac cells with such doses of morphine (0.1 or 1.0 µM) showed a significant increase in TMRE fluorescence intensity compared to the control group, indicating that morphine could prevent oxidant stress-induced mitochondrial damage. However, this protective effect of morphine was reversed by the ROS scavenger N-(2-mercaptopropionyl) glycine (MPG), suggesting that ROS accounts for the protective action of morphine. In support, morphine increased ROS generation significantly. Meanwhile, the protective effect of morphine was reversed both by the nonselective RTKs inhibitor genistein and the selective EGFRTK inhibitor AG1478, but not by the selective IGF-1RTK inhibitor AG1024, indicating that EGFRTK is involved in the protective effect of morphine. In addition, the effect of morphine on ROS generation was also reversed by genistein and AG1478, indicating that morphine produces ROS via EGFRTK. It has been reported that morphine activates Akt via ROS. Therefore, it is likely that morphine generates ROS via EGFRTK which then increases the activity of Akt. Based on the results of western blot and immunofluorescence assay, morphine markedly increased Akt phosphorylation which was blocked by AG1478. Conclusions these findings demonstrate that morphine prevents oxidant stress-induced mitochondrial damage in cardiac H9c2 muscle cells via an EGFRTK-ROS-Akt signalling pathway.