The usage and costs of national drug price-negotiated anticancer medicines in a first-tier city in Northeast China: a study based on health insurance data.

BACKGROUND: The National Drug Price Negotiation (NDPN) policy has entered a normalisation stage, aiming to alleviate, to some extent, the disease-related and economic burdens experienced by cancer patients. This study analysed the use and subsequent burden of anticancer medicines among cancer patients in a first-tier city in northeast China. METHODS: We assessed the usage of 64 negotiated anticancer medicines using the data on the actual drug deployment situation, the frequency of medical insurance claims and actual medication costs. The affordability of these medicines was measured using the catastrophic health expenditure (CHE) incidence and intensity of occurrence. Finally, we used the defined daily doses (DDDs) and defined daily doses cost (DDDc) as indicators to evaluate the actual use of these medicines in the region. RESULTS: During the study period, 63 of the 64 medicines were readily available. From the perspective of drug usage, the frequency of medical insurance claims for negotiated anticancer medicines and medication costs showed an increasing trend from 2018 to 2021. Cancer patients typically sought medical treatment at tertiary hospitals and purchased medicines at community pharmacies. The overall quantity and cost of medications for patients covered by the Urban Employee Basic Medical Insurance (UEBMI) were five times higher than those covered by the Urban and Rural Resident Medical Insurance (URRMI). The frequency of medical insurance claims and medication costs were highest for lung and breast cancer patients. Furthermore, from 2018 to 2021, CHE incidence showed a decreasing trend (2.85-1.60%) under urban patients' payment capability level, but an increasing trend (11.94%-18.42) under rural patients' payment capability level. The average occurrence intensities for urban (0.55-1.26 times) and rural (1.27-1.74 times) patients showed an increasing trend. From the perspective of drug utilisation, the overall DDD of negotiated anticancer medicines showed an increasing trend, while the DDDc exhibited a decreasing trend. CONCLUSION: This study demonstrates that access to drugs for urban cancer patients has improved. However, patients' medical behaviours are affected by some factors such as hospital level and type of medical insurance. In the future, the Chinese Department of Health Insurance Management should further improve its work in promoting the fairness of medical resource distribution and strengthen its supervision of the nation's health insurance funds.

Probucol-induced hERG Channel Reduction can be Rescued by Matrine and Oxymatrine in vitro.

BACKGROUND: The human ether-a-go-go-related gene (hERG) potassium channel is the rapidly activating component of cardiac delayed rectifier potassium current (IKr), which is a crucial determinant of cardiac repolarization. The reduction of hERG current is commonly believed to cause Long QT Syndrome (LQTs). Probucol, a cholesterol-lowering drug, induces LQTs by inhibiting the expression of the hERG channel. Unfortunately, there is currently no effective therapeutic method to rescue probucol-induced LQTs. METHODS: Patch-clamp recording techniques were used to detect the action potential duration (APD) and current of hERG. Western blot was performed to measure the expression levels of proteins. RESULTS: In this study, we demonstrated that 1 µM matrine and oxymatrine could rescue the hERG current and hERG surface expression inhibited by probucol. In addition, matrine and oxymatrine significantly shortened the prolonged action potential duration induced by probucol in neonatal cardiac myocytes. We proposed a novel mechanism underlying the probucol induced decrease in the expression of transcription factor Specificity protein 1 (Sp1), which is an established transactivator of the hERG gene. We also demonstrated that matrine and oxymatrine were able to upregulate Sp1 expression which may be one of the possible mechanisms by which matrine and oxymatrine rescued probucol-induced hERG channel deficiency. CONCLUSION: Our current results demonstrate that matrine and oxymatrine could rescue probucol-induced hERG deficiency in vitro, which may lead to potentially effective therapeutic drugs for treating acquired LQT2 by probucol in the future.

Intracellular Mechanism of Rosuvastatin-Induced Decrease in Mature hERG Protein Expression on Membrane.

The hERG potassium channel (IKr) encoded by human ether-a-go-go-related gene plays an important role in cardiac repolarization. Decreased IKr may lead to long QT syndrome, which subsequently causes torsade de pointes and sudden cardiac death. Previous studies have shown that statins inhibit IKr and are more potent in inhibiting hERG currents when combined with other drugs. Since chemical structure of rosuvastatin is similar to that of several IKr blockers (ibutilide and E-4031), the present study aimed to reveal the mechanism that underlies rosuvastatin-induced hERG current reduction and to evaluate the possibility of cardiac toxicity. The results showed that rosuvastatin reduced hERG currents by accelerating the inactivation and prolonged action potential duration (APD) in hiPSC-CMs. Meanwhile, it was observed that rosuvastatin reduced the expression of the mature hERG. Transcription factor Sp1 was involved in hERG protein downregulation induced by rosuvastatin, and the result was verified by Sp1 siRNA and Sp1 agonist epicatechin. These results indicated that rosuvastatin could potentially inhibit transcription and reduce hERG mRNA expression. The interaction between hERG and heat shock protein was evaluated to study the mechanism of trafficking inhibition through co-immunoprecipitation. We found that rosuvastatin reduces the interaction of heat shock protein 70 (Hsp70) with the hERG protein, thereby affecting the folding of the hERG channel. Additionally, rosuvastatin significantly activates ATF6, which plays a key role in the activation of the unfolded protein response (UPR) pathway. Increased expression of the molecular chaperone calnexin and calreticulin, which are activated by ATF6 to help channel folding, further confirmed UPR activation. Meanwhile, the degradation of the hERG channel was mediated by lysosomes and proteasomes. In conclusion, Rosuvastatin reduced the expression of hERG plasma membrane by two pathways, the first is to disrupt the transport of immature hERG channels to the membrane, and the second is to increase the degradation of mature hERG channels. In addition, Rosuvastatin potently blocked hERG current, delayed cardiac repolarization, and thereby prolonged APDs and QTc intervals. Therefore, caution should be taken when rosuvastatin is used in the treatment of hyperlipidemia, especially when combined with drugs that can prolong the QT interval.

TGF-ß1 pathway affects the protein expression of many signaling pathways, markers of liver cancer stem cells, cytokeratins, and TERT in liver cancer HepG2 cells.

Liver cancer is one of the most common human malignancies, and transforming growth factor-beta (TGF-ß) pathway plays a key role in its pathogenesis. To study the relationship between TGF-ß pathway and the related protein expression of many signaling pathway, markers of stem cells, CK family, and others, liver cancer HepG2 cells were transfected with siRNA directed against TGF-ß1 or were treated with exogenous TGF-ß1. Then, these protein levels were measured by Western blotting. After siRNA transfection, TGF-ß1 protein level was decreased, indicating that the siRNA against it was effective. In exogenous TGF-ß1 group, the expression of smad4, smad2/3, and ß-catenin proteins was increased, whereas that of p-smad2/3, CD133, cleaved Notch1, and epithelial cell adhesion molecule (EpCAM) proteins at 48 h was decreased. The expression of CK8 and CK18 proteins was increased at 24 h and was decreased at 48 and 96 h. In TGF-ß1-silenced group, the expression of smad2/3, ß-catenin, cleaved-notch1, and CK18 proteins was decreased, while that of smad4, p-smad2/3, CD133, EpCAM, and CK8 proteins was increased. TERT protein expression was slightly increased in exogenous TGF-ß1 group at 48 h and in TGF-ß1-silenced group at 96 h. TGF-ß1 did not affect the protein expression of CK19 and HIF-1. Thus, TGF-ß1 pathway plays an important role in cell regulation of liver cancer through the modulation of these proteins. These data will contribute to the understanding of the pathogenesis of liver cancer and the role of TGF-ß pathway in this process.

Increased expression of urotensin II is associated with poor prognosis in hepatocellular carcinoma.

Urotensin II (UII) and the urotensin II receptor (UT) exhibit mitogenic effects on tumor growth. Our previous study demonstrated that the UII/UT system is upregulated in hepatocellular carcinoma (HCC) and may enhance the proliferation of human hepatoma cells. However, the clinical significance of UII/UT expression in HCC remains unclear. The present study assessed UII messenger RNA (mRNA) expression in 129 surgical specimens obtained from HCC patients using reverse transcription quantitative-polymerase chain reaction. The association between UII mRNA expression and clinicopathological parameters and overall survival rates was also investigated. The results revealed that UII and UT mRNA expression was significantly increased in HCC tissue compared with adjacent non-cancerous liver tissue (P<0.001). Furthermore, a significant correlation was identified between UII expression and histological differentiation (P<0.01), tumor size (P<0.01) and tumor stage (P=0.026). Kaplan-Meier survival analysis indicated that overall survival time was significantly shorter in patients with high UII expression, compared with those with low UII expression (P<0.001). Multivariate analyses indicated that UII expression was an independent predictor of overall survival (odds ratio, 1.12; P<0.001). In addition, UII mRNA was correlated with vascular endothelial growth factor mRNA expression. Therefore, UII expression is an independent biomarker for the prognosis of patients with HCC and thus, the UII/UT system may present a novel therapeutic target for the treatment of HCC.

Mechanisms underlying probucol-induced hERG-channel deficiency.

The hERG gene encodes the pore-forming α-subunit of the rapidly activating delayed rectifier potassium channel (I Kr), which is important for cardiac repolarization. Reduction of I hERG due to genetic mutations or drug interferences causes long QT syndrome, leading to life-threatening cardiac arrhythmias (torsades de pointes) or sudden death. Probucol is a cholesterol-lowering drug that could reduce hERG current by decreasing plasma membrane hERG protein expression and eventually cause long QT syndrome. Here, we investigated the mechanisms of probucol effects on I hERG and hERG-channel expression. Our data demonstrated that probucol reduces SGK1 expression, known as SGK isoform, in a concentration-dependent manner, resulting in downregulation of phosphorylated E3 ubiquitin ligase Nedd4-2 expression, but not the total level of Nedd4-2. As a result, the hERG protein reduces, due to the enhanced ubiquitination level. On the contrary, carbachol could enhance the phosphorylation level of Nedd4-2 as an alternative to SGK1, and thus rescue the ubiquitin-mediated degradation of hERG channels caused by probucol. These discoveries provide a novel mechanism of probucol-induced hERG-channel deficiency, and imply that carbachol or its analog may serve as potential therapeutic compounds for the handling of probucol cardiotoxicity.

High Glucose Represses hERG K+ Channel Expression through Trafficking Inhibition.

BACKGROUND/AIMS: Abnormal QT prolongation is the most prominent cardiac electrical disturbance in patients with diabetes mellitus (DM). It is well known that the human ether-ago-go-related gene (hERG) controls the rapid delayed rectifier K+ current (IKr) in cardiac cells. The expression of the hERG channel is severely down-regulated in diabetic hearts, and this down-regulation is a critical contributor to the slowing of repolarization and QT prolongation. However, the intracellular mechanisms underlying the diabetes-induced hERG deficiency remain unknown. METHODS: The expression of the hERG channel was assessed via western blot analysis, and the hERG current was detected with a patch-clamp technique. RESULTS: The results of our study revealed that the expression of the hERG protein and the hERG current were substantially decreased in high-glucose-treated hERG-HEK cells. Moreover, we demonstrated that the high-glucose-mediated damage to the hERG channel depended on the down-regulation of protein levels but not the alteration of channel kinetics. These discoveries indicated that high glucose likely disrupted hERG channel trafficking. From the western blot and immunoprecipitation analyses, we found that high glucose induced trafficking inhibition through an effect on the expression of Hsp90 and its interaction with hERG. Furthermore, the high-glucose-induced inhibition of hERG channel trafficking could activate the unfolded protein response (UPR) by up-regulating the expression levels of activating transcription factor-6 (ATF-6) and the ER chaperone protein calnexin. In addition, we demonstrated that 100 nM insulin up-regulated the expression of the hERG channel and rescued the hERG channel repression caused by high glucose. CONCLUSION: The results of our study provide the first evidence of a high-glucose-induced hERG channel deficiency resulting from the inhibition of channel trafficking. Furthermore, insulin promotes the expression of the hERG channel and ameliorates the high-glucose-induced inhibition of the hERG channel.

The enhancement of cardiac toxicity by concomitant administration of Berberine and macrolides.

As is well-known, hERG plays an essential role in phase III repolarization of cardiac action potentials. Blocking of hERG channels can lead to LQTS. Inhibition of the metabolism of CYPs activities may elevate plasma levels, to further increase accumulation of drug on cardiac. The elevated serum levels may however elicit unexpected toxicities. Therefore, the inhibition tests of hERG and CYP are central to the preclinical studies because they may lead to severe cardiac toxicity. Berberine is widely used as an antibacterial agent and often combined with macrolides to treat gastropathy. Our objective was to assess cardiac toxicity during the combined use of Berberine with macrolides. (1) Azithromycin reduced hERG currents by accelerated channel inactivation. (2) The combination of Berberine with Azithromycin reduced hERG currents, producing an inhibitive effect stronger than use of a single drug alone, due to the high binding affinity for the onset of inactivation. (3) When cells were perfused concomitantly with Berberine and Clarithromycin, they showed a stronger inhibitive effect on hERG currents by decreasing the time constant for the onset of inactivation. (4) The combined administration of Berberine with Clarithromycin had a powerful inhibitive effect on CYP3A activities than use of a single drug alone. Collectively, these results demonstrated that concomitant use of Berberine with macrolides may require close monitoring because of potential drug toxicities, especially cardiac toxicity.

Up-regulation of miR-21 and miR-23a Contributes to As2 O3 -induced hERG Channel Deficiency.

Arsenic trioxide (As2O3) is used to treat acute pro-myelocytic leukaemia. However, the cardiotoxicity of long QT syndrome restricts its clinical application. Previous studies showed that As2O3 can damage the hERG current via disturbing its trafficking to cellular membrane. Consistent with these findings, in this study, we reported that As2O3 inhibited hERG channel at both protein and mRNA levels and damaged hERG current but did not affect channel kinetics. Further, we demonstrated that As2O3 up-regulated miR-21 and miR-23a expression in hERG-HEK293 cells and neonatal cardiomyocytes. In addition, knock-down of miR-21 by its specific antisense molecules AMO-21 was able to rescue Sp1 and hERG inhibition caused by As2O3. Consistently, phosphorylation of NF-κB, the upstream regulatory factor of miR-21, was significantly up-regulated by As2O3 . This finding revealed that regulation of the NF-κB-miR-21-Sp1 signalling pathway is a novel mechanism for As2O3-induced hERG inhibition. Meanwhile, the expression of Hsp90 and hERG was rescued by transfection with AMO-23a. And the hERG channel inhibition induced by As2O3 was rescued after being transfected with AMO-23a, which may be a molecular mechanism for the role of As2O3 in hERG trafficking deficiency. In brief, our study revealed that miR-21 and miR-23a are involved in As2O3-induced hERG deficiency at transcriptional and transportational levels. This discovery may provide a novel mechanism of As2O3-induced hERG channel deficiency, and these miRNAs may serve as potential therapeutic targets for the handling of As2O3 cardiotoxicity.

Translational toxicology and rescue strategies of the hERG channel dysfunction: biochemical and molecular mechanistic aspects.

The human ether-à-go-go related gene (hERG) potassium channel is an obligatory anti-target for drug development on account of its essential role in cardiac repolarization and its close association with arrhythmia. Diverse drugs have been removed from the market owing to their inhibitory activity on the hERG channel and their contribution to acquired long QT syndrome (LQTS). Moreover, mutations that cause hERG channel dysfunction may induce congenital LQTS. Recently, an increasing number of biochemical and molecular mechanisms underlying hERG-associated LQTS have been reported. In fact, numerous potential biochemical and molecular rescue strategies are hidden within the biogenesis and regulating network. So far, rescue strategies of hERG channel dysfunction and LQTS mainly include activators, blockers, and molecules that interfere with specific links and other mechanisms. The aim of this review is to discuss the rescue strategies based on hERG channel toxicology from the biochemical and molecular perspectives.

The rescuable function and mechanism of resveratrol on As2O3-induced hERG K⁺ channel deficiency.

Arsenic trioxide (As2O3) is used to treat acute promyelocytic leukemia. However, the cardiotoxicity of long QT syndrome restricts its clinical application. Previous studies showed that As2O3 can damage the human ether-a-go-go-related gene (hERG) current via disturbing its trafficking to cellular membrane. This study aimed to investigate whether the As2O3-insulted hERG channel can be rescued by resveratrol, a recognized cardioprotective agent. The whole-cell patch clamp technique was used to record the hERG current and action potential duration. Co-immunoprecipitation and Western blot assay were applied to determine the function of hERG-Hsp70/Hsp90 chaperone complexes and the expression alteration of protein-folding-related proteins, respectively. Compared with treatment of As2O3 alone, co-treatment with resveratrol successfully restored the current and surface expression of hERG and obviously shortened action potential duration in guinea pig ventricular myocytes. Further experiments demonstrate that resveratrol relieved As2O3-caused endoplasmic reticulum (ER) stress by restoring the function of hERG-Hsp70/Hsp90 chaperone complexes and downregulating the protein expression of ER chaperone proteins (calnexin and calreticulin) and activating transcription factor 6. In conclusion, resveratrol was able to rescue the trafficking deficiency and relieve the ER stress (ERS). Our findings suggest that resveratrol has a potential effect to alleviate the adverse effect of As2O3 on cardiotoxicity.

Ginkgo biloba extract and ginkgolide antiarrhythmic potential by targeting hERG and ICa-L channel.

We investigated the effects of Ginkgo biloba extract (GBE) and ginkgolide (GLD) on human ether-a-go-go-related gene (hERG)-encoded K(+) channels and its underlying mechanisms in the hERG-HEK293 cell line by determining GBE- and GLD-induced changes in action potential duration (APD), L-type calcium currents (ICa-L), and the intracellular calcium concentration ([Ca(2+)]i) in guinea-pig ventricular myocytes. hERG currents, APD and ICa-L were recorded using the whole-cell patch clamp technique, the [Ca(2+)]i was examined by an immunofluorescence experiment. In the present study, we found that a low concentration of GBE (0.005 mg/ml) increased hERG currents, but the high concentration of GBE (from 0.05 to 0.25 mg/ml) reduced hERG currents. GLD reduced hERG currents in a concentration-dependent manner (from 0.005 to 0.25 mg/ml). Both GBE and GLD altered kinetics of the hERG channel. GBE accelerated the activation of hERG channels without changing the inactivation curve, but reduced the time constant of inactivation; GLD did not shift the activation or the inactivation curve, but only reduced the time constant of inactivation. Both GBE and GLD shortened the APD, inhibited the ICa-L currents, and decreased the [Ca(2+)]i in isolated guinea-pig ventricular myocytes. The results indicate that GBE and GLD can prevent ischemic arrhythmias and have an antiarrhythmic effect potential via inhibition of IKr and ICa-L currents.

Primary pulmonary artery myxoma: a rare case.

Pulmonary embolism is the most frequent diagnosis for a filling defect in the pulmonary artery, but a tumor in the arteriae pulmonalis should be contained in the differential diagnosis. Primary pulmonary artery myxoma is extremely rare, and only a few cases have been reported. The early diagnosis of this disease is difficult, but it is feasible with modern radiographic methods, which play an important role in the presentation of the origin and extension of the tumor. Here, we review one case with computed tomographic (CT) and pulmonary CT angiographic findings to emphasize the significance of the imaging method in its diagnosis.

Comparative effects of liensinine and neferine on the human ether-a-go-go-related gene potassium channel and pharmacological activity analysis.

Liensinine and neferine, a kind of isoquinoline alkaloid, can antagonize the ventricular arrhythmias. The human ether-a-go-go-related gene (hERG) is involved in repolarization of cardiac action potential. We investigated the effects of liensinine and neferine on the biophysical properties of hERG channel and the underlying structure-activity relationships. The effects of liensinine and neferine were examined on the hERG channels in the stable transfected HEK293 cells using a whole-cell patch clamp technique, western blot analysis and immunofluorescence experiment. The pharmacokinetics and tissue distribution determination of liensinine and neferine in rats were determined by a validated RP-HPLC method. Liensinine and neferine induced decrease of current amplitude in dose-dependent. Liensinine reduced hERG tail current from 70.3±6.3 pA/pF in control group to 56.7±2.8 pA/pF in the 1 µM group, 53.0±2.3 pA/pF (3 µM) and 17.8±0.7 pA/pF (30 µM); the corresponding current densities of neferine-treated cells were 41.9±3.1 pA/pF, 32.3±3.1 pA/pF and 16.2±0.6 pA/pF, respectively. Neferine had binding affinity for the open and inactivated state of hERG channel, liensinine only bound to the open state. The inhibitory effects of liensinine and neferine on hERG current were attenuated in the F656V or Y652A mutant channels. Neferine distributed more quickly than liensinine in rats, which was found to be in higher concentration than liensinine. Both liensinine and neferine had no effect on the generation and expression of hERG channels. In conclusion, neferine is a more potent blocker of hERG channels than liensinine at low concentration (<10 µM), which may be due to higher hydrophobic nature of neferine compared with liensinine. Neferine may be safety even for long-term treatment as an antiarrhythmic drug.

The liposomal daunorubicin plus tamoxifen: improving the stability, uptake, and biodistribution of carriers.

The synergistic effects of tamoxifen on the sensitivity of MCF-7 cells to daunorubicin have been reported. Whether the effects of daunorubicin on MCF-7/adr cells can be improved by tamoxifen in liposomes and how tamoxifen changes daunorubicin's behavior in vivo remains unclear. The aim of this study was to investigate the effects of tamoxifen on the uptake and biodistribution of daunorubicin liposomes by breast-cancer-resistant MCF-7/adr cells in vitro and in vivo. The uptake of liposomes by MCF-7/adr cells in vitro studies was measured using flow cytometry and laser confocal microscopy. The biodistributions of carriers and free drugs were evaluated by DiR dye using in vivo imaging. Tamoxifen obviously enhanced the cellular uptake of liposomes by MCF-7/adr cells in time-dependent manners. According to the results from in vivo imaging analysis, the mean fluorescence intensity of DiR liposomes with tamoxifen in the tumor regions of MCF-7/adr tumor-bearing nude mice was much stronger than that of DiR liposomes alone (16,450 ± 1,331 versus 3,666 ± 321; n = 3). Pegylated liposomes elongated the existence of daunorubicin in the circulatory system and the enhanced permeability and retention effect enhanced its concentration in local tumor tissues, which may provide the precondition for tamoxifen further promoting the uptake by MCF-7/Adr cells in vivo. Using daunorubicin liposomes and tamoxifen together generates better biodistribution profiles in tumor tissue than using daunorubicin liposomes only, which contributes to improving the therapeutic effect of breast cancer treatment.

[Effect of the Wnt/LRP5/ß-catenin signaling pathway on the pathogenesis of postmenopausal osteoporosis].

OBJECTIVE: To investigate the role of the Wnt/LRP5/ß-catenin signaling pathway in the pathogenesis of postmenopausal osteoporosis. METHODS: Fifty female Wistar rats aged 6-month-old, were randomly divided into control group (NS, n = 24) and ovariectomized group (NOVX, n = 26), NOVX underwent bilateral ovariectomy. At 0, 4 and 8 weeks, all of rats were measured blood estrogen (E(2)) and bone mineral density (BMD), 4 and 8 weeks, low density lipoprotein receptor-related protein 5 (LRP5), ß-catenin and Runx2 mRNA in bone were measured respectively by reverse transcription (RT)-PCR. RESULTS: In 4 and 8 weeks, compared with NS which had (117 ± 29) and (114 ± 15) pmol/L in E(2) level, (0.098 ± 0.016) and (0.095 ± 0.028) g/cm(2) in BMD, NOVX had significantly decreased to (92 ± 15) and (95 ± 22) pmol/L in E(2) level (P < 0.05), (0.076 ± 0.016) and (0.052 ± 0.013) g/cm(2) in BMD values (P < 0.01). And bone tissue LRP5, ß-catenin and Runx2 mRNA expression was 1.02 ± 0.06, 1.04 ± 0.05, 1.07 ± 0.21 in NS, NOVX was significantly reduced to 0.97 ± 0.04, 0.58 ± 0.05, 0.86 ± 0.03 (P < 0.05). CONCLUSION: Wnt/LRP5/ß-catenin signaling pathway may be important in the pathogenesis of postmenopausal osteoporosis.

Impaired PI3K/Akt signal pathway and hepatocellular injury in high-fat fed rats.

AIM: to determine whether mitochondrial dysfunction resulting from high-fat diet is related to impairment of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt, also known as PKB) pathway. METHODS: rat models of nonalcoholic fatty liver were established by high-fat diet feeding. The expression of total and phosphorylated P13K and Akt proteins in hepatocytes was determined by Western blotting. Degree of fat accumulation in liver was measured by hepatic triglyceride. Mitochondrial number and size were determined using quantitative morphometric analysis under transmission electron microscope. The permeability of the outer mitochondrial membrane was assessed by determining the potential gradient across this membrane. RESULTS: after Wistar rats were fed with high-fat diet for 16 wk, their hepatocytes displayed an accumulation of fat (103.1 ± 12.6 vs 421.5 ± 19.7, P < 0.01), deformed mitochondria (9.0% ± 4.3% vs 83.0% ± 10.9%, P < 0.05), and a reduction in the mitochondrial membrane potential (389.385% ± 18.612% vs 249.121% ± 13.526%, P < 0.05). In addition, the expression of the phosphorylated P13K and Akt proteins in hepatocytes was reduced, as was the expression of the anti-apoptotic protein Bcl-2, while expression of the pro-apoptotic protein caspase-3 was increased. When animals were treated with pharmacological inhibitors of P13K or Akt, instead of high-fat diet, a similar pattern of hepatocellular fat accumulation, mitochondrial impairment, and change in the levels of PI3K, Akt, Bcl-2 was observed. CONCLUSION: high-fat diet appears to inhibit the PI3K/Akt signaling pathway, which may lead to hepatocellular injury through activation of the mitochondrial membrane pathway of apoptosis.

The novel mechanism of oxymatrine affecting HERG currents at different temperatures.

BACKGROUND/AIMS: Human ether-à-go-go-related gene (hERG) has an important role in the repolarization of the cardiac action potential. Our studies were to investigate the effects of oxymatrine (one of the natural constituents extracted from Chinese herb Sophora flavescens Ait) on hERG-encoded K(+) channels at different temperatures and its underlying mechanism. METHODS: The effects of oxymatrine were examined on hERG channels stably expressed in HEK293 cells using a whole-cell patch clamp technique. RESULTS: At the temperature 30°C, oxymatrine inhibited hERG current in a concentration-dependent manner and the IC(50) was ∼665 µM. However at the temperature of 20°C, low concentration oxymatrine C≤100 µM increased hERG current density. However, high concentration oxymatrine C>100 µM inhibited the hERG current density significantly. Oxymatrine only affected the activation kinetic of hERG channels at all temperatures and had a high binding affinity for open state of hERG channels except the 300 µM-20°C group which had a high binding affinity for inactive state of hERG channels. CONCLUSION: Oxymatrine is a low potency blocker of hERG K+ channels at 30°C, low concentration oxymatrine affect the hERG activation gating with accelerating hERG tail current at 20°C, oxymatrine is a potential hERG activator at low temperatures.

Effect of ANEPIII, a novel recombinant neurotoxic polypeptide, on sodium channels in primary cultured rat hippocampal and cortical neurons.

Previous studies have shown that the recombinant neurotoxic polypeptide BmK ANEP (ANEPIII) displayed good anti-neuroexcitation activity as demonstrated by pharmacological tests of the blockade of chemical-induced convulsive seizures. In order to search for further anticonvulsant mechanism of action of ANEPIII, the effects of ANEPIII on sodium channels were assessed using the whole-cell patch clamp recordings in primary cultures of rat hippocampal and cortical neurons. ANEPIII decreased the sodium currents in a voltage-dependent manner, which appeared as a shift of the current-voltage relation to positive potentials. The effect was reversible after washing. The concentration-responsiveness measured in hippocampal and cortical neurons revealed an IC(50) value of 124.6 nM and 192.7 nM, respectively. Furthermore, ANEPIII 1000 nM significantly shifted the activation curves of sodium current in hippocampal and cortical neurons to more positive potentials and the recovery from inactivation of sodium current was significantly slower. Voltage-dependent inactivation curves of sodium channels in hippocampal and cortical neurons did not change in the presence of 1000 nM ANEPIII. Thus, our results demonstrated that ANEPIII in submicromolar concentrations was a voltage-dependent, reversible blocker of sodium current in hippocampal and cortical neurons. It is concluded that these phenomena may explain, at least in part, the anti-neuroexciting properties of this peptide.

Blockade of HERG K+ channel by isoquinoline alkaloid neferine in the stable transfected HEK293 cells.

We studied the effects of isoquinoline alkaloid neferine (Nef) extracted from the seed embryo of Nelumbo nucifera Gaertn on Human ether-à-go-go-related gene (HERG) channels stably expressed in human embryonic kidney (HEK293) cells using whole-cell patch clamp technique, western blot analysis and immunofluorescence experiment. Nef induced a concentration-dependent decrease in current amplitude according to the voltage steps and tail currents of HERG with an IC(50) of 7.419 microM (n(H) -0.5563). Nef shifted the activation curve in a significantly negative direction and accelerated recovery from inactivation and onset of inactivation, however, slowed deactivation. In addition, it had no significant influence on steady-state inactivation curve. Western blot and immunofluorescence results suggested Nef had no significant effect on the expression of HERG protein. In summary, Nef can block HERG K(+) channels that functions by changing the channel activation and inactivation kinetics. Nef has no effect on the generation and trafficking of HERG protein. A blocked-off HERG channel was one mechanism of the anti-arrhythmic effects by Nef.