Magnesium lithospermate B dilates mesenteric arteries by activating BKca currents and contracts arteries by inhibiting Kv currents

Aim:

To examine the involvement of K⁺ channels and endothelium in the vascular effects of magnesium lithospermate B (MLB), a hydrophilic active component of Salviae miltiorrhiza Radix.

Methods:

Isolated rat mesenteric artery rings were employed to investigate the effects of MLB on KCl- or norepinephrine-induced contractions. Conventional whole-cell patch-clamp technique was used to study the effects of MLB on K⁺ currents in single isolated mesenteric artery myocytes.

Results:

MLB produced a concentration-dependent relaxation in mesenteric artery rings precontracted by norepinephrine (1 μmol/L) with an EC₅₀ of 111.3 μmol/L. MLB-induced relaxation was reduced in denuded artery rings with an EC₅₀ of 224.4 μmol/L. MLB caused contractions in KCl-precontracted artery rings in the presence of N-nitro-L-arginine methyl ester (L-NAME) with a maximal value of 130.3%. The vasodilatory effect of MLB was inhibited by tetraethylammonium (TEA) in both intact and denuded artery rings. In single smooth muscle cells, MLB activated BK_Ca currents (EC₅₀ 156.3 μmol/L) but inhibited K_V currents (IC₅₀ 26.1 μmol/L) in a voltage- and concentration-dependent manner.

Conclusion:

MLB dilated arteries by activating BK_Ca channels in smooth muscle cells and increasing NO release from endothelium, but it also contracted arteries precontracted with KCl in the presence of L-NAME.     

Metergoline inhibits the neuronal Navl.2 voltage- dependent Na^＋ channels expressed in Xenopus oocytes

Aim： Metergoline is an ergot-derived psychoactive drug that acts as a ligand for serotonin and dopamine receptors. The aim of this study was to investigate the regulatory effects of metergoline on the neuronal Nav1.2 voltage-dependent Na^＋ channels in vitro. Methods： Xenopus oocytes were injected with cRNAs encoding rat brain Nav1.2 α and β1 subunits. Voltage-activated Na^＋ currents were recorded using two-electrode voltage clamp technique. Drugs were applied though perfusion. Results： Both metergoline and lidocaine reversibly and concentration-dependently inhibited the peak of Na^＋ currents with IC50 values of 3.6±4.2 and 916.9±98.8 μmol/L, respectively. Metergoline （3 pmol/L） caused a 6.8±1.2 mV depolarizing shift of the steady-state activation curve of the Na^＋ currents, and did not alter the inactivation curve. In contrast, lidocaine （3 μmol/L） caused a 12.7±1.2 mV hyperpolarizing shift of the inactivation curve of the Na^＋ currents without changing the steady-state activation curve. Both metergoline and lidocaine produced tonic and use-dependent inhibition on the peak of Na^＋ currents. Conclusion： Metergoline exerts potent inhibition on the activity of neuronal Nav1.2 channels, which may contribute to its actions on the central nervous system.     

Vasorelaxant and antihypertensive effects of formononetin through endothelium-dependent and -independent mechanisms

Aim:

To investigate the mechanisms underlying the vasorelaxant effect of formononetin, an O-methylated isoflavone, in isolated arteries, and its antihypertensive activity in vivo.

Methods:

Arterial rings of superior mesenteric arteries, renal arteries, cerebral basilar arteries, coronary arteries and abdominal aortas were prepared from SD rats. Isometric tension of the arterial rings was recorded using a myograph system. Arterial pressure was measured using tail-cuff method in spontaneously hypertensive rats.

Results:

Formononetin (1–300 μmol/L) elicited relaxation in arteries of the five regions that were pre-contracted by KCl (60 mmol/L), U46619 (1 μmol/L) or phenylephrine (10 μmol/L). The formononetin-induced relaxation was reduced by removal of endothelium or by pretreatment with L-NAME (100 μmol/L). Under conditions of endothelium denudation, formononetin (10, 30, and 100 μmol/L) inhibited the contraction induced by KCl and that induced by CaCl₂ in Ca²⁺-free depolarized medium. In the absence of extracellular Ca²⁺, formononetin (10, 30, and 100 μmol/L) depressed the constriction caused by phenylephrine (10 μmol/L), but did not inhibit the tonic contraction in response to the addition of CaCl₂ (2 mmol/L). The contraction caused by caffeine (30 mmol/L) was not inhibited by formononetin (100 μmol/L). Formononetin (10 and 100 μmol/L) reduced the change rate of Ca²⁺-fluorescence intensity in response to KCl (50 mmol/L). In spontaneously hypertensive rats, formononetin (5, 10, and 20 mg/kg) slowly lowered the systolic, diastolic and mean arterial pressure.

Conclusion:

Formononetin causes vasodilatation via two pathways: (1) endothelium-independent pathway, probably due to inhibition of voltage-dependent Ca²⁺ channels and intracellular Ca²⁺ release; and (2) endothelium-dependent pathway by releasing NO. Both the pathways may contribute to its antihypertensive effect.     

18β-Glycyrrhetinic acid preferentially blocks late Na current generated by ΔKPQ Nav1.5 channels

Aim:

To compare the effects of two stereoisomeric forms of glycyrrhetinic acid on different components of Na⁺ current, HERG and Kv1.5 channel currents.

Methods:

Wild-type (WT) and long QT syndrome type 3 (LQT-3) mutant ΔKPQ Nav1.5 channels, as well as HERG and Kv1.5 channels were expressed in Xenopus oocytes. In addition, isolated human atrial myocytes were used. Two-microelectrode voltage-clamp technique was used to record the voltage-activated currents.

Results:

Superfusion of 18β-glycyrrhetinic acid (18β-GA, 1–100 μmol/L) blocked both the peak current (I_Na,P) and late current (I_Na,L) generated by WT and ΔKPQ Nav1.5 channels in a concentration-dependent manner, while 18α-glycyrrhetinic acid (18α-GA) at the same concentrations had no effects. 18β-GA preferentially blocked I_Na,L (IC₅₀=37.2±14.4 μmol/L) to I_Na,P (IC₅₀=100.4±11.2 μmol/L) generated by ΔKPQ Nav1.5 channels. In human atrial myocytes, 18β-GA (30 μmol/L) inhibited 47% of I_Na,P and 87% of I_Na,L induced by Anemonia sulcata toxin (ATX-II, 30 nmol/L). Superfusion of 18β-GA (100 μmol/L) had no effects on HERG and Kv1.5 channel currents.

Conclusion:

18β-GA preferentially blocked the late Na current without affecting HERG and Kv1.5 channels.     

Establishment and characterization of a new drug surviving cell line Am1010, derived directly from muscle metastases of a human lung adenocarcinoma patient with multi-drug-resistance tOcisplatin, taxol, and gefitinib

Aim:

To Characterize a new human lung cancer cell line Am1010, derived from drug-surviving cells (DSCs).

Methods:

The Am1010 cell line was established after 4 cycles of chemotherapy from an arm muscle metastasic tumor of a patient diagnosed with lung adenocarcinoma. The cell line has been remained in continuous culture for more than one year during this study.

Results:

The Am1010 cell line demonstrated in vitro multi-drug-resistance to cisplatin, taxol, and gefitinib. The Am1010 cell doubling time without drug treatment was 42.395 h. The IC₅₀ value of cisplatin was 4.299 μmol/L and >10 μmol/L for the Am1010 and P0318 (a cell line derived from non-DSCs) cells, respectively. The IC₅₀ value of taxol was 0.067 μmol/L and >1 μmol/L for the Am1010 and P0318 cells, respectively. The IC₅₀ value of gefitinib was 15.233 μmol/L and >70 μmol/L for Am1010 and P0318 cells, respectively. 11 genes involved in the focal adhesion and cell adhesion pathways were found to be differentially expressed. The cells of Am1010 have a significantly larger chromosome number than most lung cancer cell lines.

Conclusion:

This novel DSCs derived lung cancer cell line will be a valuable in vitro tool for the investigation of lung cancer drug resistance and metastasis.     

Protective effect of Bu-7, a flavonoid extracted from Clausena lansium, against rotenone injury in PC12 cells

Aim:

To investigate the protective effect and underlying mechanisms of Bu-7, a flavonoid isolated from the leaves of Clausena lansium, against rotenone-induced injury in PC12 cells.

Methods:

The cell viability was evaluated using MTT assay. The cell apoptosis rate was analyzed using flow cytometry. JC-1 staining was used to detect the mitochondrial membrane potential (MMP). Western blotting analysis was used to determine the phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (p38), tumor protein 53 (p53), Bcl-2–associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), and caspase 3.

Results:

Treatment of PC12 cells with rotenone (1–20 μmol/L) significantly reduced the cell viability in a concentration-dependent manner. Pretreatment with Bu-7 (0.1 and 10 μmol/L) prevented PC12 cells from rotenone injury, whereas Bu-7 (1 μmol/L) had no significant effect. Pretreatment with Bu-7 (0.1 and 10 μmol/L) decreased rotenone-induced apoptosis, attenuated rotenone-induced mitochondrial potential reduction and suppressed rotenone-induced protein phosphorylation and expression, whereas Bu-7 (1 μmol/L) did not cause similar effects. Bu-7 showed inverted bell-shaped dose-response relationship in all the effects.

Conclusion:

Bu-7 protects PC12 cells against rotenone injury, which may be attributed to MAP kinase cascade (JNK and p38) signaling pathway. Thus, Bu-7 may be a potential bioactive compound for the treatment of Parkinson''s disease.     

Effects of diltiazem and propafenone on the inactivation and recovery kinetics of fKv1.4 channel currents expressed in Xenopus oocytes

Aim:

To investigate the effects of diltiazem, an L-type calcium channel blocker, and propafenone, a sodium channel blocker, on the inactivation and recovery kinetics of fKv1.4, a potassium channel that generates the cardiac transient outward potassium current.

Methods:

The cRNA for fKv1.4ΔN, an N-terminal deleted mutant of the ferret Kv1.4 potassium channel, was injected into Xenopus oocytes to express the fKv1.4ΔN channel in these cells. Currents were recorded using a two electrode voltage clamp technique.

Results:

Diltiazem (10 to 1000 μmol/L) inhibited the fKv1.4ΔN channel in a frequency-dependent, voltage-dependent, and concentration-dependent manner, suggesting an open channel block. The IC₅₀ was 241.04±23.06 μmol/L for the fKv1.4ΔN channel (at +50 mV), and propafenone (10 to 500 μmol/L) showed a similar effect (IC₅₀=103.68±10.13 μmol/L). After application of diltiazem and propafenone, fKv1.4ΔN inactivation was bi-exponential, with a faster drug-induced inactivation and a slower C-type inactivation. Diltiazem increased the C-type inactivation rate and slowed recovery in fKv1.4ΔN channels. However, propafenone had no effect on either the slow inactivation time constant or the recovery.

Conclusion:

Diltiazem and propafenone accelerate the inactivation of the Kv1.4ΔN channel by binding to the open state of the channel. Unlike propafenone, diltiazem slows the recovery of the Kv1.4ΔN channel.     

Apoptosis induced by genipin in human leukemia K562 cells: involvement of c-Jun N-terminal kinase in G₂/M arrest

Aim:

To investigate the effect of genipin on apoptosis in human leukemia K562 cells in vitro and elucidate the underlying mechanisms.

Methods:

The effect of genipin on K562 cell viability was measured using trypan blue dye exclusion and cell counting. Morphological changes were detected using phase-contrast microscopy. Apoptosis was analyzed using DNA ladder, propidium iodide (PI)-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of genipin on cell cycle distribution was determined using PI staining. Caspase 3 activity was analyzed to detect apoptosis at different time points. Protein levels of phospho-c-Jun, phosphor-c-Jun N-terminal kinase (p-JNK), phosphor-p38, Fas-L, p63, and Bax and the release of cytochrome c were detected using Western blot analysis.

Results:

Genipin reduced the viability of K562 cells with an IC₅₀ value of approximately 250 μmol/L. Genipin 200–400 μmol/L induced formation of typical apoptotic bodies and DNA fragmentation. Additionally, genipin 400 μmol/L significantly increased the caspase 3 activity from 8–24 h and arrested the cells in the G₂/M phase. After stimulation with genipin 500 μmol/L, the levels of p-JNK, p-c-Jun, Fas-L, Bax, and cytochrome c were remarkably upregulated, but there were no obvious changes of p-p38. Genipin 200–500 μmol/L significantly upregulated the Fas-L expression and downregulated p63 expression. Dicoumarol 100 μmol/L, a JNK1/2 inhibitor, markedly suppressed the formation of apoptotic bodies and JNK activation induced by genipin 400 μmol/L.

Conclusion:

These results suggest that genipin inhibits the proliferation of K562 cells and induces apoptosis through the activation of JNK and induction of the Fas ligand.     

Novel KCNQ2 channel activators discovered using fluorescence-based and automated patch-clamp-based high-throughput screening techniques

Aim:

To establish an improved, high-throughput screening techniques for identifying novel KCNQ2 channel activators.

Methods:

KCNQ2 channels were stably expressed in CHO cells (KCNQ2 cells). Thallium flux assay was used for primary screening, and 384-well automated patch-clamp IonWorks Barracuda was used for hit validation. Two validated activators were characterized using a conventional patch-clamp recording technique.

Results:

From a collection of 80 000 compounds, the primary screening revealed a total of 565 compounds that potentiated the fluorescence signals in thallium flux assay by more than 150%. When the 565 hits were examined in IonWorks Barracuda, 38 compounds significantly enhanced the outward currents recorded in KCNQ2 cells, and were confirmed as KCNQ2 activators. In the conventional patch-clamp recordings, two validated activators ZG1732 and ZG2083 enhanced KCNQ2 currents with EC50 values of 1.04±0.18 μmol/L and 1.37±0.06 μmol/L, respectively.

Conclusion:

The combination of thallium flux assay and IonWorks Barracuda assay is an efficient high-throughput screening (HTS) route for discovering KCNQ2 activators.     

Wogonin ameliorates lipotoxicity-induced apoptosis of cultured vascular smooth muscle cells via interfering with DAG-PKC pathway

Aim:

To investigate the effects of wogonin (5,7-dihydroxy-8-methoxyflavone) extracted from Scutellaria baicalensis Georgi (S baicalensis) on lipotoxicity-induced apoptosis of vascular smooth muscle cells (VSMCs) and the underlying mechanisms.

Methods:

Cultured VSMCs were used. Apoptosis of VSMCs was induced by palmitate (0.75 mmol/L), and detected using TUNEL assay. The expression levels of protein and phosphorylated protein were measured using Western blot analysis.

Results:

Treatment of VSMCs with wogonin (10, 25 and 50 μmol/L) significantly attenuated the apoptosis and endoplasmic reticulum (ER) stress induced by palmitate in concentration- and time-dependent manners. Wogonin (50 μmol/L) decreased palmitate-induced reactive oxygen species (ROS) generation. The ER stress inhibitor 4-phenyl butyric acid (5 mmol/L) significantly decreased palmitate-induced apoptotic cells, and occluded the anti-apoptotic effect of wogonin (25 μmol/L). Wogonin (10, 25 and 50 μmol/L) significantly reduced the intracellular diacylglycerol (DAG) accumulation and expression levels of phosphorylated PKCs in palmitate-treated VSMCs.

Conclusion:

Our results suggest that wogonin inhibits lipotoxicity-induced apoptosis of VSMCs via suppressing the intracellular DAG accumulation and subsequent inhibition of PKC phosphorylation. Wogonin has therapeutic potential for the prevention and treatment of atherosclerosis.     

Application of headspace solid phase microextraction for study of noncovalent interaction of borneol with human serum albumin

Aim:

To investigate noncovalent interactions between borneol and human serum albumin (HSA) under near-physiological conditions.

Methods:

A 65-μm polydimethylsiloxane (PDMS) fiber was selected for sampling. The extraction temperature was kept at 37 °C, and the extraction time was optimized at 10 min. Borneol solutions of different concentrations were equilibrated in 600 μmol/L HSA and 67 mmol/L phosphate buffer solution (pH 7.4, 37 °C) for 24 h prior to solid phase microextraction (SPME) using headspace mode. The binding properties were obtained based on the calculation of extracted borneol amount using gas chromatography (GC) determination.

Results:

The headspace SPME extraction method avoided disturbance from the HSA binding matrix. The recovery showed good linearity for the borneol concentrations over the range of 0.4–16.3 μmol/L with a regression coefficient (R²) of 0.9998. The limit of detection and lower limit of quantitation were determined to be 0.01 μmol/L and 0.4 μmol/L, respectively. The binding constant and the percentage binding rate were estimated to be 2.4×10³(mol/L)^-1 and 59.5%, respectively.

Conclusion:

Headspace SPME coupled to GC is a simple, sensitive and rapid method for the study of borneol binding to HSA. The method may be applied in the determination of other protein binding properties in human plasma.     

Bezafibrate enhances proliferation and differentiation of osteoblastic MC3T3-E1 cells via AMPK and eNOS activation

Aim:

To investigate the effects of bezafibrate on the proliferation and differentiation of osteoblastic MC3T3-E1 cells, and to determine the signaling pathway underlying the effects.

Methods:

MC3T3-E1 cells, a mouse osteoblastic cell line, were used. Cell viability and proliferation were examined using MTT assay and colorimetric BrdU incorporation assay, respectively. NO production was evaluated using the Griess reagent. The mRNA expression of ALP, collagen I, osteocalcin, BMP-2, and Runx-2 was measured using real-time PCR. Western blot analysis was used to detect the expression of AMPK and eNOS proteins.

Results:

Bezafibrate increased the viability and proliferation of MC3T3-E1 cells in a dose- and time-dependent manner. Bezafibrate (100 μmol/L) significantly enhanced osteoblastic mineralization and expression of the differentiation markers ALP, collagen I and osteocalcin. Bezafibrate (100 μmol/L) increased phosphorylation of AMPK and eNOS, which led to an increase of NO production by 4.08-fold, and upregulating BMP-2 and Runx-2 mRNA expression. These effects could be blocked by AMPK inhibitor compound C (5 μmol/L), or the PPARβ inhibitor GSK0660 (0.5 μmol/L), but not by the PPARα inhibitor MK886 (10 μmol/L). Furthermore, GSK0660, compound C, or N^G-nitro-L-arginine methyl ester hydrochloride (L-NAME, 1 mmol/L) could reverse the stimulatory effects of bezafibrate (100 μmol/L) on osteoblast proliferation and differentiation, whereas MK886 only inhibited bezafibrate-induced osteoblast proliferation.

Conclusion:

Bezafibrate stimulates proliferation and differentiation of MC3T3-E1 cells, mainly via a PPARβ-dependent mechanism. The drug might be beneficial for osteoporosis by promoting bone formation.     

Involvement of Bcl-2, Src, and ERα in gossypol-mediated growth inhibition and apoptosis in human uterine leiomyoma and myometrial cells

Aim:

To investigate the effect of gossypol on the growth of cultured human uterine leiomyoma and myometrial cells, the level of Bcl-2 and the activity of Src and estrogen receptor (ERα).

Methods:

Human uterine leiomyoma and adjacent normal myometrial cells were cultured in vitro. Both cell types were treated with a graded concentration of gossypol. Cell viability was assayed using CCK-8. Morphological change was observed with optical and electronic microscopy. Apoptosis was evaluated using TUNEL assay. Levels of Bcl-2, ERα and Src were analyzed using Western blotting.

Results:

Gossypol significantly inhibited growth and promoted apoptosis in cultured human uterine leiomyoma cells with the IC₅₀ value and its corresponding 95% confidence intervals (CI) of 6.5 (4.0–10.5), 9.0 (4.9–16.5), and 7.5 (4.0–14.1) μmol/L at 20, 40, and 60 h, respectively. Gossypol exerted inhibitory effects on the myometrial cells with the IC₅₀ value and its 95% CI of 49.1 (28.3–85.0), 14.5 (7.7–27.4), and 2.6 (1.2–5.6) μmol/L at 20, 40, and 60 h, respectively. Compared with control, gossypol 0.1-3.0 μmol/L markedly decreased the protein expression of Bcl-2 (P<0.05) in both leiomyoma and myometrial cells in a concentration-dependent manner, and significantly suppressed the level of phospho-Tyr416Src (P<0.05) in both cell types at 3.0 μmol/L without obvious alteration of c-Src and phospho-Tyr527Src levels (P>0.05). In addition, gossypol markedly reduced both the expression of ERα (P<0.05) at the low concentration of 0.1 μmol/L in the myometrial cells and the level of phospho-ser167ERα (P<0.05) at the high concentration of 3.0 μmol/L in the leiomyoma cells.

Conclusion:

Gossypol inhibits proliferation and induces apoptosis in human uterine leiomyoma and myometrial cells. It is likely that the mechanisms of action involve reducing the protein level of Bcl-2 and the activity of Src and ERα.     

Effects of neferine on Kv4.3 channels expressed in HEK293 cells and ex vivo electrophysiology of rabbit hearts

Aim:

Neferine is an isoquinoline alkaloid isolated from seed embryos of Nelumbo nucifera (Gaertn), which has a variety of biological activities. In this study we examined the effects of neferine on Kv4.3 channels, a major contributor to the transient outward current (I_to) in rabbit heart, and on ex vivo electrophysiology of rabbit hearts.

Methods:

Whole-cell Kv4.3 currents were recorded in HEK293 cells expressing human cardiac Kv4.3 channels using patch-clamp technique. Arterially perfused wedges of rabbit left ventricles (LV) were prepared, and transmembrane action potentials were simultaneously recorded from epicardial (Epi) and endocardial (Endo) sites with floating microelectrodes together with transmural electrocardiography (ECG).

Results:

Neferine (0.1–100 μmol/L) dose-dependently and reversibly inhibited Kv4.3 currents (the IC₅₀ value was 8.437 μmol/L, and the maximal inhibition at 100 μmol/L was 44.12%). Neferine (10 μmol/L) caused a positive shift of the steady-state activation curve of Kv4.3 currents, and a negative shift of the steady-state inactivation curve. Furthermore, neferine (10 μmol/L) accelerated the inactivation but not the activation of Kv4.3 currents, and markedly slowed the recovery of Kv4.3 currents from inactivation. Neferine-induced blocking of Kv4.3 currents was frequency-dependent. In arterially perfused wedges of rabbit LV, neferine (1, 3, and 10 μmol/L) dose-dependently prolonged the QT intervals and action potential durations (APD) at both Epi and Endo sites, and caused dramatic increase of APD₁₀ at Epi sites.

Conclusion:

Neferine inhibits Kv4.3 channels likely by blocking the open state and inactivating state channels, which contributes to neferine-induced dramatic increase of APD₁₀ at Epi sites of rabbit heart.     

Discovery of a retigabine derivative that inhibits KCNQ2 potassium channels

Aim:

Retigabine, an activator of KCNQ2-5 channels, is currently used to treat partial-onset seizures. The aim of this study was to explore the possibility that structure modification of retigabine could lead to novel inhibitors of KCNQ2 channels, which were valuable tools for KCNQ channel studies.

Methods:

A series of retigabine derivatives was designed and synthesized. KCNQ2 channels were expressed in CHO cells. KCNQ2 currents were recorded using whole-cell voltage clamp technique. Test compound in extracellular solution was delivered to the recorded cell using an ALA 8 Channel Solution Exchange System.

Results:

A total of 23 retigabine derivatives (HN31-HN410) were synthesized and tested electrophysiologically. Among the compounds, HN38 was the most potent inhibitor of KCNQ2 channels (its IC₅₀ value=0.10±0.05 μmol/L), and was 7-fold more potent than the classical KCNQ inhibitor XE991. Further analysis revealed that HN38 (3 μmol/L) had no detectable effect on channel activation, but accelerated deactivation at hyperpolarizing voltages. In contrast, XE991 (3 μmol/L) did not affect the kinetics of channel activation and deactivation.

Conclusion:

The retigabine derivative HN38 is a potent KCNQ2 inhibitor, which differs from XE991 in its influence on the channel kinetics. Our study provides a new strategy for the design and development of potent KCNQ2 channel inhibitors.     

Telmisartan attenuates isoproterenol-induced cardiac remodeling in rats via regulation of cardiac adiponectin expression

Aim:

To investigate whether telmisartan (Telm) pretreatment attenuates isoproterenol (Iso)-induced postinfarction remodeling (PIR) in rats, and whether the effect of Telm is associated with cardiac expression of adiponectin.

Methods:

PIR was induced in male Wistar rats with two consecutive injections of Iso (80 mg/kg, sc) at an interval of 24 h. Primary culture of ventricular myocytes from neonatal rats was prepared. Iso-induced cardiomyocyte injury was assessed based on cell growth and lactate dehydrogenase (LDH) activity. Cardiac adiponectin expression was measured using qRT-PCR and immunoblot analysis.

Results:

In the rats with PIR, Telm (10 mg·kg⁻¹·d⁻¹, po for 65 d) suppressed Iso-induced increases in gravimetric parameters, cardiomyocyte diameter and collagen volume fraction, but had no effect on Iso-induced myocardial hypertrophy and interstitial fibrosis. The protective effect of Telm was associated with enhanced protein expression of cardiac adiponectin. In cultured cardiomyocytes, Telm (5–20 μmol/L) inhibited the cell death and LDH release induced by Iso (10 μmol/L), and reversed Iso-induced reduction in adiponectin protein expression. In cardiomyocytes exposed to Iso (20 μmol/L), GW9662 (30 μmol/L), a selective antagonist of PPAR-γ, blocked the effects of Telm pretreatment on adiponectin protein expression, as well as the protective effects of Telm on Iso-induced cell injury.

Conclusion:

Telm attenuates Iso-induced cardiac remodeling and cell injury, which is associated with induction of cardiac adiponectin expression.     

Quercetin sensitizes human glioblastoma cells to temozolomide in vitro via inhibition of Hsp27

Aim： Quercetin is an effective Hsp27 inhibitor and has been reported to facilitate tumor cell apoptosis. The aim of this study was to investigate whether quercetin could sensitize human glioblastoma cells to temozolomide （TMZ） in vitro.
Methods： Both U251 and U87 human glioblastoma cells were treated with quercetin and/or TMZ for 48 h. Cell viability was detected using the MTT assay. Cell apoptosis was analyzed with caspase-3 activity kits and flow cytometry. Hsp27 expression and phosphorylation were examined using Western blot analysis. RNA interference using Hsp27 siRNA oligos was performed to knock down the gene expression of Hsp27.
Results： TMZ （200 or 400 μmol/L） alone effectively inhibited the viability of U251 and U87 cells. When combined with quercetin （30 μmol/L）, TMZ （100 μmol/L） significantly inhibited the cell viability, and the inhibition of TMZ （200 and 400 μmol/L） was enhanced. TMZ or quercetin anole did not affect caspase-3 activity and cell apoptosis, while TMZ combined with quercetin significantly increased caspase-3 activity and induced cell apoptosis. TMZ anole significantly increased Hsp27 phosphorylation in U251 and U87 cells, while quercetin or Hsp27 siRNA oligos combined with TMZ attenuated TMZ-induced Hsp27 phosphorylation and significantly inhibited Hsp27 expression.
Conclusion： Combined treatment with TMZ and quercetin efficiently suppressed human glioblastoma cell survival in vitro.     

Salvianolic acid A inhibits angiotensin II-induced proliferation of human umbilical vein endothelial cells by attenuating the production of ROS

Aim:

To investigate the action of salvianolic acid A (SalA) on angiotensin II (Ang II)-induced proliferation of human umbilical vein endothelial cells (HUVECs) and the possible signaling pathways mediating this action.

Methods:

Cell proliferation was examined with MTT assay. The expression levels of Src phosphorylation (phospho-Src), Akt phosphorylation (phospho-Akt), and NADPH oxidase 4 (Nox4) in HUVECs were determined by Western blot. The production of reactive oxygen species (ROS) was estimated using fluorescence-activated cell sorting (FACS).

Results:

SalA (6.25–50 μmol/L) did not affect the viability of HUVECs. Treatment of HUVECs with Ang II (1 μmol/L) markedly increased the cell viability; pretreatment of HUVECs with SalA (12.5, 25 and 50 μmol/L) prevented Ang II-induced increase of the cell viability in a concentration-dependent manner. Treatment of HUVECs with Ang II (1 μmol/L) markedly up-regulated the protein expression levels of phospho-Src, phospho-Akt (473) and Nox4; pretreatment of HUVECs with SalA (12.5, 25 and 50 μmol/L) blocked all the effects in a concentration-dependent manner. Treatment of HUVECs with Ang II (1 μmol/L) dramatically increased ROS production in HUVECs; pretreatment of HUVECs with SalA (12.5, 25 and 50 μmol/L) blocked the ROS production in a concentration-dependent manner.

Conclusion:

SalA inhibits Ang II-induced proliferation of HUVECs via reducing the expression levels of phospho-Src and phospho-Akt (473), thereby attenuating the production of ROS.     

Magnesium sulfate enhances non-depolarizing muscle relaxant vecuronium action at adult muscle-type nicotinic acetylcholine receptor in vitro

Aim:

To investigate the effect of magnesium sulfate and its interaction with the non-depolarizing muscle relaxant vecuronium at adult muscle-type acetylcholine receptors in vitro.

Methods:

Adult muscle-type acetylcholine receptors were expressed in HEK293 cells. Drug-containing solution was applied via a gravity-driven perfusion system. The inward currents were activated by brief application of acetylcholine (ACh), and recorded using whole-cell voltage-clamp technique.

Results:

Magnesium sulfate (1–100 mmol/L) inhibited the inward currents induced ACh (10 μmol/L) in a concentration-dependent manner (IC₅₀=29.2 mmol/L). The inhibition of magnesium sulfate was non-competitive. In contrast, vecuronium produced a potent inhibition on the adult muscle-type acetylcholine receptor (IC₅₀=8.7 nmol/L) by competitive antagonism. Magnesium sulfate at the concentrations of 1, 3, and 6 mmol/L markedly enhanced the inhibition of vecuronium (10 nmol/L) on adult muscle-type acetylcholine receptors.

Conclusion:

Clinical enhancement of vecuronium-induced muscle relaxation by magnesium sulfate can be attributed partly to synergism between magnesium sulfate and non-depolarizing muscle relaxants at adult muscle-type acetylcholine receptors.