Polar Molecules Regulating the Regio- and Stereoselectivity of Polymerization of Conjugated Dienes Catalyzed by CGC-Type Rare-Earth Metal Catalysts.

Herein, a concise, effective, and scalable strategy is reported that the introduction of polar molecules (PMs) (e.g., anisole (PhOMe), phenetole (PhOEt), 2-methoxynaphthalene (NaphOMe), thioanisole (PhSMe), and N,N-dimethylaniline (PhNMe2)) as continuously coordinated neutral ligand of cationic active species in situ generated from the constrain-geometry-configuration-type rare-earth metal complexes A-F/AliBu3/[Ph3C][B(C6F5)4] ternary systems can easily switch the regio- and stereoselectivity of the polymerization of conjugated dienes (CDs, including 2-subsituted CDs such as isoprene (IP) and myrcene (MY), 1,2-disubstituted CD ocimene (OC), and 1-substituted polar CD 1-(para-methoxyphenyl)-1,3-butadiene (p-MOPB)) from poor selectivities to high selectivities (for IP and MY: 3,4-selectivity up to 99%; for OC: trans-1,2-selectivity up to 93% (mm up to 90%); for p-MOPB: 3,4-syndioselectivity (3,4- up to 99%, rrrr up to 96%)). DFT calculations explain the continuous coordination roles of PMs on the regulation of the regio- and stereoselectivity of the polymerization of CDs. In comparison with the traditional strategies, this strategy by adding some common PMs is easier and more convenient, decreasing the synthetic cost and complex operation of new metal catalyst and cocatalyst. Such regio- and stereoselective regulation method by using PMs is not reported for the coordination polymerization of olefins catalyzed by rare-earth metal and early transition metal complexes.

Dexmedetomidine Confers Protection Against Neuronal Oxygen Glucose Deprivation-Reperfusion by Regulating SIRT3 Mediated Autophagy.

Dexmedetomidine (Dex) plays protective effects on brain ischemia-reperfusion (I/R) injury, but its mechanism remains unclear. In this study, we aimed to investigate whether Dex protects neurons against I/R injury by activating SIRT3 mediated autophagy. The oxygen glucose deprivation-reperfusion (OGD/R) model was constructed in HT22 cells. Different doses of Dex (50 ng/mL, 100 ng/mL and 500 ng/mL) were treated to observe the changes of autophagy and SIRT3 expression. Further, the mimic of SIRT3 and SIRT3 inhibitor were used to analyze the effects of Dex on the SIRT3 expression in HT22 cells. Additionally, the autophagy inhibitor and AMPK inhibitor were used to analyze the effects of Dex on SIRT3 mediated autophagy. The cells viability, oxidative stress and ATP were observed using assay kits. The mitochondrial membrane potential (MMP) and death were analyzed by flow cytometry. The degree of autophagy was observed by acridine orange staining. Western blotting was used to analyze the expression of autophagy related proteins and AMPK/mTOR pathway related proteins. After Dex treatment, the OGD/R induced cell injury was significantly improved through decreasing the levels of LDH and H2O2, increasing levels of ATP and MMP. Furthermore, Dex increased the degree of autophagy and expression of SIRT3 in OGD/R injured cells. Through overexpression of SIRT3, the OGD/R induced cell injury was also clearly improved. But the SIRT3 inhibitor or autophagy inhibitor covered the roles of Dex. Additionally, AMPK inhibitor played an opposite role compared with the effects of Dex treatment. From this study, the protection mechanism of Dex on neurons I/R injury might related to the activation of SIRT3 mediated autophagy.

Bepridil, a class IV antiarrhythmic agent, can block the TREK-1 potassium channel.

BACKGROUND: The TWIK-related potassium channel (TREK-1) can be regulated by different stimuli. However, it is not clear whether some antiarrhythmics affect the activity of TREK-1. In the present study, the effect of bepridil on the TREK-1 currents is investigated. METHODS: In a TREK-1 stably-expressed HEK-293 cell line (HEK-TREK-1), U251MG cells, and isolated mouse ventricular myocytes, the TREK-1 current and action potentials were recorded by the patch-clamp technique. The standard voltage protocol was a 200 ms constant potential at 20 mV, followed bya 500 ms ramp from -90 to +20 mV (HEK-TREK-1) or +80 mV (U251MG cells and myocytes) every 10 s. The currents at +20 mV or +80 mV were used for analysis. The docking study of bepridil's binding model in the TREK-1 channel was performed using the Swissdock web service. RESULTS: In HEK-TREK-1 cells, BL1249 induced a significantly large outwardly rectifying current with similar baseline TREK-1 current characteristic, with a reversal potential (-70 mV). The concentration of half-maximal activation (EC50) of BL1249 was 3.45 µM. However, bepridil decreased the baseline TREK-1 currents, with a concentration of half-maximal inhibition (IC50) 0.59 µM and a Hill coefficient of 1.1. Also, bepridil inhibited BL1249-activated TREK-1 currents, with an IC50 4.08 µM and a Hill coefficient of 3.22. The outside-out patch-clamp confirmed bepridil inhibited BL1249-activated TREK-1 currents. In U251MG cells and myocytes, BL1249 activated outwardly rectifying endogenous TREK-1 currents, which could be inhibited by bepridil. BL1249 (10 µM) could decrease the peak value and reduce the duration of the action potential. Bepridil (10 µM) prolonged the duration of action potential of myocytes. The docking study revealed that bepridil might affect the K+ pore domain and the M4 modulator pocket. CONCLUSIONS: Bepridil may be a blocker for the TREK-1K+channel at a clinically therapeutic concentration, providing a new mechanism of TREK-1 regulation and bepridil's antiarrhythmic effect.

Sevoflurane suppresses migration and invasion of glioma cells by regulating miR-146b-5p and MMP16.

Background: Glioma is the most common brain tumor with poor prognosis all over the world. Anesthetics have been demonstrated to have important impacts on cell migration and invasion in different cancers. However, the underlying mechanism that allows anesthetics-mediated progression of glioma cells remains elusive. Methods: Sevoflurane (Sev), a class of common anesthetics, was used to expose to U87-MG and U251 cells. The expressions of microRNA-146b-5p (miR-146b-5p) and matrix metallopeptidase 16 (MMP16)were measured by quantitative real-time polymerase chain reaction or western blot. Transfection was performed in glioma cells with miR-146b-5p inhibitor, inhibitor negative control, MMP16 overexpression vector, empty vector, small interfering RNA against MMP16 or scramble. Cell migration and invasion were analyzed by the trans-well assay. The interaction between miR-146b-5p and MMP16 was explored by luciferase activity and RNA immunoprecipitation assays. Results: Sev treatment inhibited migration and invasion of glioma cells. The expression of miR-146b-5p was enhanced and MMP16 protein was decreased in glioma cells after exposure of Sev. Knockdown of miR-146b-5p or overexpression of MMP16 reversed Sev-induced inhibition of migration and invasion of glioma cells. Moreover, MMP16 was indicated as a target of miR-146b-5p and its silencing attenuated the regulatory role of miR-146b-5p abrogationin Sev-treated glioma cells. Conclusion: Sev impeded cell migration and invasion through regulating miR-146b-5p and MMP16 in glioma, indicating a novel theories foundation for the application of anesthetics like Sev in glioma.

Upregulated TRPC5 plays an important role in development of nasal polyps by activating eosinophilic inflammation and NF-κB signaling pathways.

The pathophysiology of nasal polyps (NP) remains unclear, however, several ion channels may participate. Whether transient receptor potential canonical (TRPC) channel play a role in NP remains unknown. We investigated expression of TRPC, eosinophil infiltration, IL-6, and NF-κB in 58 patients with NP and 35 control subjects using hematoxylin-eosin (HE) staining, immunohistochemistry, real-time fluorescence quantitative reverse transcription PCR (real-time RT-PCR), Western blotting, and calcium imaging. Compared with normal nasal mucosa, TRPC5 mRNA and protein expression increased in NP. Eosinophil counts, IL-6 expression, and phosphorylation levels of NF-κB were higher in NP than in normal mucosa. TRPC5 expression was positively correlated with eosinophils, IL-6, and phosphorylation levels of NF-κB. Blocking of TRPC5 channel decreased store-operated calcium influx, IL-6 expression, and phosphorylation levels of NF-κB in blood eosinophils from patients with NP. In conclusion, TRPC5 was upregulated in NP and played an important role in development of NP by activating eosinophilic inflammation and NF-κB signal pathways.

Lipophilic but not hydrophilic statin functionally inhibit volume-activated chloride channels by inhibiting NADPH oxidase in monocytes.

Volume-activated Cl- channels (VACCs) can be activated by hypotonic solutions and have been identified in many cell types. Here, we investigated the effects of different statins on VACCs in monocytes. Whole-cell patch clamp recordings demonstrated that a hypotonic solution induced 5-nitro-2- (3-phenylpropylamino) benzoic acid (NPPB)- and 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS)-sensitive VACC currents in human peripheral monocytes and RAW 264.7 cells. The VACC currents were inhibited by the lipophilic statin (simvastatin) but not by the hydrophilic simvastatin acid and pravastatin. A low-molecular-weight superoxide anion scavenger (tiron, 1 mM) and inhibitor of NADPH oxidase (DPI 10 µM) was able to abolish the VACC currents. A hypotonic solution increased the reactive oxygen species (ROS) detected by the fluorescence of dichlorodihydrofluorescein (DCF), which was abolished by tiron and DPI. NPPB, DIDS, and simvastatin but not pravastatin decreased the fluorescence of DCF. Simvastatin could not further decrease VACC currents when pretreated with tiron or DPI, whereas exogenous H2O2 (100 µM), increased the VACC currents and overcame the blockade of VACC currents by simvastatin. Functionally, hypotonic solution increased the TNF-α mRNA expression, which could be decreased by tiron, DPI, NPPB, DIDS and simvastatin but not pravastatin. However, simvastatin could not decrease the TNF-α expression further when pretreatment with tiron, DPI, NPPB or DIDS. We conclude that lipophilic (simvastatin) rather than hydrophilic statin inhibit VACCs and decrease hyposmolality induced inflammation in monocytes by inhibiting NADPH oxidase.

Glibenclamide decreases ATP-induced intracellular calcium transient elevation via inhibiting reactive oxygen species and mitochondrial activity in macrophages.

Increasing evidence has revealed that glibenclamide has a wide range of anti-inflammatory effects. However, it is unclear whether glibenclamide can affect the resting and adenosine triphosphate (ATP)-induced intracellular calcium ([Ca(2+)]i) handling in Raw 264.7 macrophages. In the present study, [Ca(2+)]i transient, reactive oxygen species (ROS) and mitochondrial activity were measured by the high-speed TILLvisION digital imaging system using the indicators of Fura 2-am, DCFDA and rhodamine-123, respectively. We found that glibenclamide, pinacidil and other unselective K(+) channel blockers had no effect on the resting [Ca(2+)]i of Raw 264.7 cells. Extracellular ATP (100 µM) induced [Ca(2+)]i transient elevation independent of extracellular Ca(2+). The transient elevation was inhibited by an ROS scavenger (tiron) and mitochondria inhibitor (rotenone). Glibenclamide and 5-hydroxydecanoate (5-HD) also decreased ATP-induced [Ca(2+)]i transient elevation, but pinacidil and other unselective K(+) channel blockers had no effect. Glibenclamide also decreased the peak of [Ca(2+)]i transient induced by extracellular thapsigargin (Tg, 1 µM). Furthermore, glibenclamide decreased intracellular ROS and mitochondrial activity. When pretreated with tiron and rotenone, glibenclamide could not decrease ATP, and Tg induced maximal [Ca(2+)]i transient further. We conclude that glibenclamide may inhibit ATP-induced [Ca(2+)]i transient elevation by blocking mitochondria KATP channels, resulting in decreased ROS generation and mitochondrial activity in Raw 264.7 macrophages.

Protective role of acidic pH-activated chloride channel in severe acidosis-induced contraction from the aorta of spontaneously hypertensive rats.

Severe acidic pH-activated chloride channel (ICl,acid) has been found in various mammalian cells. In the present study, we investigate whether this channel participates in reactions of the thoracic aorta to severe acidosis and whether it plays a role in hypertension. We measured isometric contraction in thoracic aorta rings from spontaneously hypertensive rats (SHRs) and normotensive Wistar rats. Severe acidosis induced contractions of both endothelium-intact and -denuded thoracic aorta rings. In Wistar rats, contractions did not differ at pH 6.4, 5.4 and 4.4. However, in SHRs, contractions were higher at pH 5.4 or 4.4 than pH 6.4, with no difference between contractions at pH 5.4 and 4.4. Nifedipine, ICl,acid blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4,4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS) inhibited severe acidosis-induced contraction of aortas at different pH levels. When blocking ICl,acid, the remnant contraction was greater at pH 4.4 than pH 5.4 and 6.4 for both SHRs and Wistar rats. With nifedipine, the remnant contraction was greatly reduced at pH 4.4 as compared with at pH 6.4 and 5.4. With NPPB or DIDS, the ratio of remnant contractions at pH 4.4 and 5.4 (R4.4/5.4) was lower for SHRs than Wistar rats (all <1). However, with nifedipine, the R4.4/5.4 was higher for SHRs than Wistar rats (both >1). Furthermore, patch clamp recordings of ICl,acid and intracellular Ca(2+) measurements in smooth muscle cells confirmed these findings. ICl,acid may protect arteries against excess vasoconstriction under extremely acidic extracellular conditions. This protective effect may be decreased in hypertension.

Extracellular acidic pH-activated, outward rectifying chloride currents can be regulated by reactive oxygen species in human THP-1 monocytes.

Extracellular acidic pH-activated chloride channels (ICl,acid) have been found in a variety of mammalian cells. In the present study, the expression and regulation of ICl,acid were investigated in THP-1 cells. Patch clamp recordings demonstrated that an extracellular acidic solution induced an outward rectified current, which could be blocked by the Cl(-) channel blocker. The currents exhibited time-dependent facilitation and inactivation. The relative anion permeability of this current followed the sequence Cl(-)>Br(-)>I(-)>gluconate. NADPH oxidase inhibitors did not decrease pH 4.4-induced currents. However, reactive oxygen species (ROS) scavengers and mitochondrial inhibitors inhibited pH 4.4-induced currents. Fluorescence imaging of intracellular ROS and mitochondrial activity confirmed these findings. We conclude that ICl,acid occurs in human THP-1 cells and that ICl,acid may be regulated by intracellular ROS mainly originating from mitochondria.

Hydrogen inhalation decreases lung graft injury in brain-dead donor rats.

BACKGROUND: The process of brain death induces acute lung injury in donors and aggravates ischemia-reperfusion injury (IRI) in grafts. Hydrogen, a new anti-oxidant, attenuates IRI in several organ transplant models. We examined whether 2% inhaled hydrogen would show favorable effects on lung grafts from brain-dead donor rats. METHODS: Brain-dead donor rats inhaled mixed gases with either 50% oxygen and 50% nitrogen or mixed gases with 2% hydrogen, 50% oxygen and 48% nitrogen for 2 hours. The recipients inhaled the same gas as the donors and were euthanized 2 hours after lung transplantation. RESULTS: Hydrogen improved PaO(2)/FIO(2) and PVO(2)/FIO(2) from the arterial and pulmonary venous blood in recipients and decreased the lung injury score in grafts from brain-dead donors. Hydrogen decreased the amount of IL-8 and TNF-α in serum, inhibited the activity of malondialdehyde and myeloperoxidase, and increased the activity of superoxide dismutase in the lung grafts from brain-dead donors. Furthermore, hydrogen decreased the apoptotic index of the cells and inhibited the protein expression of intercellular adhesion molecule-1 and caspase-3 in lung grafts from brain-dead donors. CONCLUSIONS: Hydrogen can exert protective effects on lung grafts from brain-dead donors through anti-inflammatory, anti-oxidant and anti-apoptotic mechanisms.

Overexpression of CyclinD1 and underexpression of p16 correlate with lymph node metastases in laryngeal squamous cell carcinoma in Chinese patients.

BACKGROUND: CyclinD1 and p16 are involved in the regulation of G1 checkpoint and may play an important role in the tumorgenesis of laryngeal squamous cell carcinoma (LSCC). Previous studies have examined the level of expression of cyclinD1 or p16 in LSCC but no such information is available for their relation and their correlation with lymph node metastasis in Chinese patients. PATIENTS AND METHODS: A total of 58 patients underwent surgical resection of laryngeal tumours in the Department of Otolaryngology, Qilu Hospital Shandong University between January 2001 and December 2002. All pathologic specimens were available for immunohistochemical study using antibodies against cyclinD1 and p16. RESULTS: Compared with normal epithelium, expression of CyclinD1 in the LSCC was significantly higher (62.1% vs. 10.0%, P < 0.05), expression of p16 was significantly lower (48.3% vs. 90.0%, P < 0.05); CyclinD1 expression in LSCC was up-regulated in TNM classification (r (s) = 0.409, P < 0.05) as well as with cervical lymph node metastases (r (s) = 0.294, P < 0.05); p16 expression in LSCC was down-regulated with cervical lymph node metastases (r (s) = -0.275, P < 0.05); negative significant correlation between p16 immunostaining and CyclinD1 was observed in LSCC (r (s) = -0.331, P < 0.05). CONCLUSION: There was a negative correlation between CyclinD1 expression and p16 expression in LSCC. The over-expression of CyclinD1 and the under-expression of p16 may play a significant role in the incidence and development of LSCC and may be important indicators for cervical lymph node metastases in Chinese patients of LSCC.

[Anti-tumor effect of chemotherapeutic drugs on human gastric cancer cells in vitro and the relationship with Bcl-2 expression].

OBJECTIVE: To evaluate in vitro anti-tumor effect of chemotherapeutic drugs on human gastric cancer cells, and investigate the relationship with Bcl-2 expression. METHODS: Single cell suspension was prepared from fresh gastric cancer tissue and exposed to taxol (Tax), 5-fluorouracil (5-FU), cisplatin (CDDP), adriamycin (ADM), mitomycin (MMC) respectively for 48 hours. Metabolic activity and inhibitory rate of cells were detected by MTT assay. Expression of Bcl-2 was examined with immunohistochemistry. RESULTS: The inhibitory rates of cancer cells exposed to chemotherapeutic drugs were different and Tax, 5-FU, CDDP had remarkably higher rates than ADM and MMC. The lower differentiated gastric cancer cells were more sensitive than the higher ones. Positive expression rate of Bcl-2 was 80% and the positive cells showed resistance to 5-FU, ADM and MMC. CONCLUSIONS: Chemosensitive testing by MTT assay can constitute the prediction for the application of chemotherapeutic drugs individually. Overexpression of Bcl-2 may contribute to multiple drug-resistance of tumors.

[Expressions of cyclin D1 and p16 in hypopharyngeal squamous cell carcinoma and their clinical significance].

OBJECTIVE: To study the expressions of Cyclin D1 and p16 proteins in hypopharyngeal squamous cell carcinoma and their clinical significance. METHOD: Immunohistochemical technology (P-V) was applied to detect the expression of Cyclin D1 and p16 in 36 cases of hypopharyngeal squamous cell carcinoma and 10 cases of normal epithelium. RESULT: (1) The expression of cyclin D1 in the tumorous cell was significantly higher than that in normal epithelium (P < 0.05). The expression of p16 in the tumorous cell was significantly lower than that in normal epithelium (P < 0.05); (2) The positive rate of Cyclin D1 was significantly correlated with clinical stage and cervical lymph node metastases (P < 0.05). The positive rate of p16 was correlated with cervical lymph node metastases (P < 0.05). The positive rates of Cyclin D1 and p16 were not correlated with the gender, age, tumor size, primary lesion and cell differentiation (P > 0.05); (3) There was correlation between the expression of Cyclin D1 and the expression of p16 (r(s) = -0.420, P < 0.05). CONCLUSION: The over-expression of Cyclin D1 and the under-expression of p16 may play a significant role in the occurrence incidence and development of hypopharyngeal squamous cell carcinoma, and may be important indicators for cervical lymph node metastases.

[Anti-tumor effects of chemotherapeutic drugs on human gastric cancer cells in vitro and the relationship with expression of hTERT mRNA].

OBJECTIVE: To evaluate in vitro antitumor effects of chemotherapeutic drugs, and investgate the relationship with expression of hTERT mRNA in human gastric cancer tissues. METHODS: Fresh samples of gastric cancer obtained from operation room were prepared to single-cell suspension (3 x 10(5) to 5 x 10(5) cells ml(-1)) and were separately exposed to taxol (TAX), cisplatin (CDDP), 5-fluorouracil (5-Fu), adriamycin (ADM), mitomycin (MMC) for 48 hours. Metabolic activity and inhibitory rate of the cells were determined by trypan blue exclusion and MTT assay. Expression of hTERT mRNA was detected by in situ hybridization (ISH). RESULTS: The inhibition rate of cancer cells exposed to chemotherapeutic drugs was different, and that of TAX, CDDP, 5-Fu was significantly higher than that of ADM and MMC. The positive rate of hTERT mRNA expression was 90.0% (54/60) and positive cells showed resistance to 5-Fu and ADM. CONCLUSION: Overexpression of hTERT mRNA may contribute to primary drug-resistance of tumors. Chemosensitivity tests by MTT assay may contribute to prediction of effectness in applying chemotherapeutic drugs and identify drug-resistant cases.