Suxiao Jiuxin Pills Prevent Ventricular Fibrillation from Inhibiting L-type Calcium Currents CaV1.2 in vivo and in vitro / 中国结合医学杂志

OBJECTIVE@#To investigate whether Suxiao Jiuxin Pills (SJP), a Chinese herbal remedy, is an anti-ventricular fibrillation (VF) agent.@*METHODS@#VF was induced by isoproterenolol (ISO) intraperitoneal injection followed by electrical pacing in mice and rabbits. The effects of SJP on the L-type calcium channel current (CaV1.2), voltage-dependent sodium channel current (INa), rapid and slow delayed rectifier potassium channel current (IKr and IKs, respectively) were studied by whole-cell patch-clamp method. Computer simulation was implemented to incorporate the experimental data of SJP effects on the CaV1.2 current into the action potential (AP) and pseudo-electrocardiography (pseudo-ECG) models.@*RESULTS@#SJP prevented VF induction and reduced VF durations significantly in mice and rabbits. Patch-clamp experiments revealed that SJP decreased the peak amplitude of the CaV1.2 current with a half maximal concentration (IC50) value of 16.9 mg/L (SJP-30 mg/L, -32.8 ± 6.1 pA; Verapamil, -16.2 ±1.8 pA; vs. control, -234.5 ±16.7 pA, P<0.01, respectively). The steady-state activation curve, inactivation curve, and the recovery from inactivation of the CaV1.2 current were not shifted significantly. Specifically, SJP did not altered INa, IKr, and IKs currents significantly (SJP vs. control, P>0.05). Computer simulation showed that SJP-reduced CaV1.2 current shortened the AP duration, transiting VF into sinus rhythm in pseudo-ECG.@*CONCLUSION@#SJP reduced VF via inhibiting the CaV1.2 current with in vivo, in vitro, and in silico studies, which provide experimental basis for SJP anti-VF clinical application.

Effect of Sodium Salicylate on Calcium Currents and Exocytosis in Cochlear Inner Hair Cells: Implications for Tinnitus Generation / 神经科学通报·英文版

Sodium salicylate is an anti-inflammatory medication with a side-effect of tinnitus. Here, we used mouse cochlear cultures to explore the effects of salicylate treatment on cochlear inner hair cells (IHCs). We found that IHCs showed significant damage after exposure to a high concentration of salicylate. Whole-cell patch clamp recordings showed that 1-5 mmol/L salicylate did not affect the exocytosis of IHCs, indicating that IHCs are not involved in tinnitus generation by enhancing their neuronal input. Instead, salicylate induced a larger peak amplitude, a more negative half-activation voltage, and a steeper slope factor of Ca2+ current. Using noise analysis of Ca2+ tail currents and qRT-PCR, we further found that salicylate increased the number of Ca2+ channels along with CaV1.3 expression. All these changes could act synergistically to enhance the Ca2+ influx into IHCs. Inhibition of intracellular Ca2+ overload significantly attenuated IHC death after 10 mmol/L salicylate treatment. These results implicate a cellular mechanism for tinnitus generation in the peripheral auditory system.

Exploration and insights into the cellular internalization and intracellular fate of amphiphilic polymeric nanocarriers

The beneficial or deleterious effects of nanomedicines emerge from their complex interactions with intracellular pathways and their subcellular fate. Moreover, the dynamic nature of plasma membrane accounts for the movement of these nanocarriers within the cell towards different organelles thereby not only influencing their pharmacokinetic and pharmacodynamic properties but also bioavailability, therapeutic efficacy and toxicity. Therefore, an in-depth understanding of underlying parameters controlling nanocarrier endocytosis and intracellular fate is essential. In order to direct nanoparticles towards specific sub-cellular organelles the physicochemical attributes of nanocarriers can be manipulated. These include particle size, shape and surface charge/chemistry. Restricting the particle size of nanocarriers below 200 nm contributes to internalization

MicroRNA-629-5p promotes osteosarcoma proliferation and migration by targeting caveolin 1

Osteosarcoma is a highly malignant tumor that occurs in the bone. Previous studies have shown that multiple microRNAs (miRNAs) regulate the development of osteosarcoma. This study aimed to explore the role of miR-629-5p and its target gene, caveolin 1 (CAV1), in osteosarcoma development. To analyze the expression of miR-629-5p and CAV1 mRNA in osteosarcoma tissues and cell lines, qRT-PCR analysis was performed. Dual-luciferase reporter experiments were subsequently performed to validate the relationship between CAV1 and miR-629-5p. CCK8 assay was used to measure osteosarcoma cell proliferation, and wound-healing assay was performed to study their migratory phenotype. Our findings revealed that miR-629-5p was overexpressed in osteosarcoma tissues and cells, and thereby enhanced cell proliferation and migration. Further, we validated that miR-629-5p targets CAV1 mRNA directly. CAV1 expression, which was negatively correlated with miR-629-5p expression, was found to be downregulated in osteosarcoma tissue samples. Moreover, our data showed that an increase in CAV1 level led to a decline in osteosarcoma cell proliferation and migration, which could be rescued by miR-629-5p upregulation. Overall, our study confirmed that miR-629-5p promoted osteosarcoma proliferation and migration by directly inhibiting CAV1.

Ginsenoside Rb1 protects brain through Cav-1 for mice with cerebral ischemia-reperfusion injury / 中国临床药理学与治疗学

AIM: To investigate the protective effect of ginsenoside Rb1 on brain through Cav-1 in mice with cerebral ischemia-reperfusion injury. METHODS: One hundred and twenty C57/B6 mice were randomly divided into sham operation group, model group, model + ginsenoside Rb1 group, ginsenoside Rb1+ Cav-1 siRNA group, ginsenoside Rb1+siNC group, 24 in each group. The model of cerebral ischemia-reperfusion injury in mice was established by middle cerebral artery occlusion (MCAO). The ginsenoside Rb1 group received intraperitoneally injection of ginsenoside Rb1 (40 mg/kg); the sham operation group and model group were intraperitoneally injected with an equal amount of physiological saline immediately after modeling. For the ginsenoside Rb1+ cav-1 siRNA group and the ginsenoside Rb1+siNC group, cav-1 siRNA and siNC were injected into the lateral ventricle 24 h before molding, respectively, and the other operations were the same as the ginsenoside Rb1 group. The neurobehavioral scores of the mice in each group were measured at 24 h after reperfusion, and the water content of brain tissue, cerebral infarction volume, Cav-1 mRNA and Cav-1, Bcl-2 and Bax protein expressions in the cerebral cortex penumbra were measured in each group. RESULTS:Compared with the sham operation group, the neurobehavioral scores, cerebral infarction volume and brain tissue water content in the model group were significantly increased (P<0.05), and the expressions of Cav-1 mRNA and Cav-1 protein, and the Bcl-2 /Bax ratio were significantly decreased (P<0.05). Compared with the model group, the neurobehavioral scores, cerebral infarction volume and brain tissue water content in the ginsenoside Rb1 group were significantly decreased, and the expressions of Cav-1 mRNA and Cav-1 protein, and the Bcl-2 /Bax ratio were significantly increased (P<0.05). Compared with the ginsenoside Rb1 group, the neurobehavioral scores, cerebral infarction volume and brain tissue water content in the ginsenoside Rb1 + cav-1 siRNA group were significantly increased, and the expressions of Cav-1 mRNA and Cav-1 protein, and the Bcl-2 /Bax ratio were significantly decreased (P<0.05). CONCLUSION: Ginsenoside Rb1 can protects brain for mice with cerebral ischemia-reperfusion injury. After Cav-1 siRNA decreased the expression of Cav-1 protein in the brain tissue of mice, it significantly reverses the cerebral protective effect of ginsenoside Rb1, indicating that Cav-1 protein mediated the cerebral protective effect of ginsenoside Rb1 on cerebral ischemia reperfusion injury mice.

Development of an Indirect ELISA Featuring Plates Coated with Column Chromatographically Purified Canine Adenovirus Type-1 Antigen

Canine adenovirus type 1 (CAV-1) causes infectious hepatitis in members of the family Canidae, including dogs. An indirect enzyme-linked immunosorbent assay (I-ELISA) that detects CAV-1 antibodies is required for large-throughput tests of dog sera. We collected 165 serum samples from dogs of Chungbuk and Gyeongbuk provinces between February 2016 and October 2018. The Korean CAV-1 vaccine strain CAV1V was propagated in Madin-Darby canine kidney (MDCK) cells and purified via Nuvia cPrime anion-exchange chromatography; the virus served as an I-ELISA antigen. Virus-neutralizing anti-CAV-1 titers in dog sera were measured using the virus neutralization (VN) method. The I-ELISA was optimized using purified CAV-1 antigen and serum samples. This kit was used to evaluate dog sera. The VN and I-ELISA data were compared. The sensitivity, specificity, and accuracy of the I-ELISA were 97.0%, 74.2%, and 92.7% compared to the VN assay, respectively. The I-ELISA data significantly correlated with those of VN (r = 0.88). These results suggest that the I-ELISA is useful for serosurveillance of CAV-1 in dog sera.

Investigation on Anti-atherosclerosis Mechanism of Tiaopi Huxin Prescription Based on Cav- 1/NF-κB Pathway / 中国药房

OBJECTIVE： To study the effects of Tiaopi huxin prescription （TPHXP） on the atherosclerosis （AS） of ApoE－/－ mice， and to investigate its mechanism. METHODS： Forty male ApoE－/－ mice were divided into blank group， model group， simvastatin group （positive control， 5 mg/kg） and TPHXP low-dose and high-dose groups （50， 150 mg/kg）， with 8 mice in each group. Except that blank group was given common diet， other groups were given high-lipid diet to induce AS model. After modeling， administration groups were given relevant medicine intragastrically， and blank group and model group were given constant volume of normal saline intragastrically， once a day， for consecutive 12 weeks. After last medication， the serum levels of TC， TG， LDL-C and HDL-C were determined by spectrophotometry. The serum level of NO was detected by nitrate reduction method. The serum levels of IL-6 and VCAM-1 were determined by ELISA. After separating thoracic aorta， HE staining was used to observe the formation of plaque in the thoracic aorta of mice in each group， and the corrected plaque area was calculated. Western blotting was conducted to determine the expression of NF-κB p65， Cav-1 and eNOS. RESULTS： Compared with blank group， the serum levels of TC， TG， LDL-C， IL-6 and VCAM-1 were increased significantly in model group， while the levels of HDL-C and NO were decreased significantly （P＜0.01）. The plaque of thoracic aorta was obvious and the corrected plaque area were increased significantly （P＜0.01）. The relative expression of NF-κB p65 and Cav-1 were increased significantly， while the relative expression of eNOS was decreased significantly （P＜0.01）. Compared with model group， the serum levels of TC， TG and LDL-C in administration groups， the serum levels of IL-6 and VCAM-1 in simvastatin group and TPHXP high-dose group were decreased significantly， while the serum levels of HDL-C and NO were increased significantly in administration groups （P＜0.05 or P＜0.01）. In administration groups， the plaques of thoracic aorta were reduced and the corrected plaque area was decreased significantly （P＜0.05 or P＜0.01）； the relative expression of NF-κB p65 and Cav-1 were decreased significantly， while the relative expression of eNOS was increased significantly （P＜0.05 or P＜0.01）. CONCLUSIONS： TPHXP can regulate the level of blood lipid， decrease the level of inflammatory factors and inhibit the formation of AS plaque， the mechanism of which may be associated with inhibiting Cav-1/NF-κB pathway.

Effects of Daurisoline on L-Type Calcium Channel CaV1.2 Expressed in HEK293 Cells / 中国药师

Objective:To investigate the effects of daurinsoline (DS) on L-type calcium channel Cav1.2 expressed in HEK293 cells.Methods:Cav1.2 was transferred into HEK293 cells using Lipofectamine 2000, and the effects of DS on Cav1.2 currents (ICav1.2) were analyzed by whole-cell patch clamp techniques. Results:DS at 1,3 and 10 μmol·L-1could inhibit the ICav1.2in HEK293 cells in a dose-dependent manner. The inhibitory rate was(14.68 ± 4.02) %,(32.37 ± 6.63) % and(59.63 ± 5.23) %,respectively. The inhibitory rate of DS at 3 μmol·L-1was 40 % of that of 3 μmol·L-1isradipine(a L-type calcium channel blocker). Conclusion:DS can inhibit the ICav1.2in HEK293 cells in a dose-dependent manner and the inhibition of DS is weaker than that of isradipine.

The effects of low intensity pulsed ultrasound on the expression of calcium transportation associated proteins during dentin-pulp complex injury and repair in rats / 实用口腔医学杂志

Objective: To investigate low intensity pulsed ultrasound (LIPUS) on the expression of L-type calcium ion channels(cav1. 2) and Na +-Ca2 + exchangers(NCX1) during dentin-pulp complex injury and repair in rats. Methods: Cavity preparation was made on the upper right first molar of 40 male adult SD rats,20 of them and the upper left first molar of the other 20 were randomly chosen for LIPUS irradiation(frequency: 1. 5 MHz,200 μs pulses,pulse repetition frequency: 1 KHz,ISATA 30 mW/cm2,20 min /d),so the animals were randomly allocated into 4 groups(n = 10): Control group,LIPUS group,cavity preparation group and cavity preparation + LIPUS group. At 1,3,7,14 d post-irradiation the rats were sacrificed respectively for HE stain and immunohistochemical analysis. Results: Reparative dentin formation was observed at 14 days after cavity preparation and LIPUS irradiation,the expression of Cav1. 2(L-type) and NCX1 in this group were increased significantly at day 1 and day 3. Compared with the control group, the expression of Cav1. 2 in LIPUS group increased at day 1 post-irradiation. Conclusion: LIPUS may enhance tertiary dentin formation and up-regulate the expression of Cav1. 2 and NCX1 at the early period of dentin injury.

Association of Polymorphisms in CAV1/CAV2 Locus with Type 2 Diabetes in Chinese Han Population / 中山大学学报(医学科学版)

[Objective]To study the association of CAV1/CAV2 gene polymorphisms with type 2 diabetes in Chinese Han population.[Methods]14 single nucleotide polymorphisms(SNPs)of CAV1/CAV2 gene were genotyped in 272 pa-tients with type 2 diabetes mellitus(T2DM group)and 287 subjects with normal glucose tolerance(control group)by ma-trix-assisted laser desorption ionization-time of flight-mass spectrometry(MALDI-TOF-MS).Waist circumference,body mass index,plasma glucose,serum insulin and lipid profiles were measured.Homeostatic model assessment of insulin resis-tance(HOMA-IR)and β-cell function(HOMA-β)were calculated.[Results]The minor allele frequency(MAF)distri-butions of CAV1 rs926198,CAV2 rs2270188,and rs1052990 were significantly different between T2DM group and con-trol group(P=0.008,0.021,and 0.045,respectively). After adjusting for age,gender,and BMI,logistic regression analysis showed that minor allele carriers(CC/CT genotype)of CAV1 rs926198 displayed a particularly increased risk of developing T2DM compared to major allele homozygotes(TT genotype)(OR=2.240,95% CI=1.415-3.544,P=0.001). GG/GA genotype carriers of CAV1 rs3807986 had lower odds for T2DM than that of AA genotype(OR=0.640,95% CI=0.449-0.913,P=0.014). Compared with TT genotype,GG/GT genotype of CAV2 rs2270188 was a protective factor for T2DM(OR=0.616,95% CI=0.432-0.878,P=0.007). Significant genotype association with T2DM was also identified in CAV2 rs1052990(GG/GT versus TT genotype:OR=0.658,95% CI=0.453-0.956,P=0.028). Multiple linear regression showed that minor allele C of SNP rs926198 was associated with an increased level of HOMA-IR(beta=1.010,P<0.001) and minor allele G of SNP rs2270188 was associated with a decreased level of HOMA-IR(beta=-0.379,P=0.023). No significant association was identified between any SNP and HOMA-β.Allele G of CAV1 rs3807986 and CAV2 rs2270188 were also associated with a decreased level of LDL-C(P=0.033 and 0.030,respectively).[Conclusion]CAV1/CAV2 locus might be the candidate genes for conferring susceptibility to T2DM in the Chinese Han population.SNP rs926198, rs3807986,rs2270188,and rs1052990 in CAV1/CAV2 locus were associated with T2DM risk perhaps through insulin resistance pathway.

The regulation and gating mechanism of Cav1.2 current by KCNE1 subunit / 中华急诊医学杂志

Objective To explore the regulation and mechanism of Cav1.2 current by KCNE1. Methods Transient transfection was used to transfer Cav1.2 channel plasmids separately or together with KCNE1 plasmids into HEK293 cells. The experiment was divided into 2 groups (15 cells in each group):Cav1.2 group, Cav1.2+KCNE1 group.The whole-cell patch clamp technique was used to record Cav1.2 current and gating dynamics. Results After co-transfection of KCNE1 with Cav1.2, Cav1.2 current decreased significantly. At 0 mV, peak current density of Cav1.2 was reduced from (-14.8±2.5) pA/pF to (-7.5±1.6) pA/pF (n=15, P<0.01). Based on the gate control mechanism, it is found that the regulation of Cav1.2 current by KCNE1 mainly makes the steady-state inactivation curve of the channel shifted to a more negative direction, thus accelerating the inactivation. Meanwhile, the recovery process of the channel after inactivation is slowed down and the recovery time constant was prolonged. Conclusions The KCNE1 subunit can reduce the current density of Cav1.2 by changing the channel inactivation and recovery process.

RalA regulates ROS and ATP production in cancer cells by association with cavelolin-1 / 医用生物力学

Objective By analyzing mitochondrial function,reactive oxygen species (ROS) and adenosine triphosphate (ATP) production under different levels of RalA and caveolin-1 (Cav-1) expression,to investigate the regulation role of RalA played in cancer metabolism and explore the possibility of its regulation role involved in Cav-1 and caveolae motility.Methods Firstly,RalA and Cav-1 expression were inhibited by siRNA in breast cancer cell line MDA-MB-231,and then the changes of mitochondrial membrane potential (MMP),ROS produc tion,ATP generation and L-lactate level before and after inhibition were assessed by Western blotting,confocal microscope and fluorescence quantification.Results (1) The decreased RalA and Cav-1 expression led to a significant reduction of MMP directly.(2) Low RalA and Cav-1 expression resulted in an inhibition of ATP production and an increase of H2O2 generation.With the reduction of MMP,mitochondrial malfunction was observed.(3) With mitochondrial function suppression,an elevated level of glycolysis metabolite L-lactate was also detected in RalA and Cav-1 deprived cells.Conclusions RalA and Cav-1 mediate cellular metabolic switch by inhibiting mitochondrial function and simultaneously boosting glycolysis.This regulation role of RalA depends on its association with Cav-1,and possibly is related to the endocytosis and motility of caveolae.The research findings enrich the cancer metabolic studies,and provide a novel approach for cancer therapeutic strategy targeted to cellular metabolism.

The Expression of Cav1.3 Calcium Channels in Adult Rat Cochlea / 听力学及言语疾病杂志

Objective To investigate the expression of Cav 1.3 calcium channel in adult rat cochlea and study its role in auditory physiology and pathology.Methods The sprague-dawley rats were used as experimental subjects.The distribution of Cav1.3 calcium channel in the cochlea was detected by immunofluorescence technique.The expression of Cav1.3 was measured with Western blot (WB) and RT-PCR.Results Immunofluorescence photographs revealed that Cav 1.3 calcium channel localized in the lateral wall membrane,hair cells,stria vascularis,spiral ganglion cell,spiral ligment,spiral prominence,and limbus laminae spiralis.The results of WB and RT-PCR inform Cav1.3 calcium channel gene (CACNA1D) were measured in the cochlea and kidney.The expression of Cav1.3was mainly in the basilar membrane.Moderate expression was observed in the spiral ganglion and stria vascularis.Conclusion The preliminary study revealed the distribution of Cav 1.3 calcium channel gene(CACNA1D)in adult rat cochle possesses tissue specificity,providing a theoretical basis for further research in auditory physiology and pathology.

Protection of SAM on KCl-injured primary cultured neurons via reducing Cav-1 expression / 中国药理学通报

Aim To investigate the protective effects of S-adenosylmethionine (SAM)on KCl-stimulated pri-mary cultured neurons and the potential mechanism. Methods The primary cultured cortical neurons were randomly divided into three groups:normal (Con) group,KCl group and SAM-KCl group. The changes of cell morphology were observed by microscope,and the mRNA and protein expression levels of caveolin-1 (Cav-1)in each group were detected by qPCR and Western blot. Results After treated with 0. 05 mol· L - 1 KCl for 12 hours,the primary cultured neurons showed morphological injury,shortening of neurites, reduction of soma mass and accumulation of cells. While neurons pretreated with 0. 02 mol·L-1 SAM for 6 hours could significantly reduce the damage induced by KCl. qPCR and Western blot analysis showed that SAM could reduce the neurons expression of Cav-1 mRNA and protein that induced by KCl. Conclusion SAM can protect neurons from the damage induced by KCl,and Cav-1 plays a critical role in this process.

Study on anti-tumor mechanism of sinomenine / 中国免疫学杂志

Objective:To investigate the anti-proliferation and anti-metastasis effects and study the molecular mechanism of sinomenine in cell line(HepG2).Methods: HepG2 cells were cultured together with different treatment concentrations of sinomen-ine.The effect of sinomenine on inhibition of growth of HepG2 cells were determined by methyl thiazolyl tetrazolium(MTT) assay.The effect of sinomenine on inhibiting metastasis of HepG2 cells were determined by Transwell assay.The inhibitory effect of sinomenine on reverse transcriptase(RT) was studied using inhibitory kinetic method,on the basis,the reactive oxygen species(ROS) of HepG2 cells was monitored by indirect fluorescent labeling.The protein expressions of CASP3,CASP9,CAV1 and SOX2 were analyzed by Western blot experiment.Results: Sinomenine inhibited the proliferation and metastasis of HepG2 cells significantly.Sinomenine had a good inhibitory effect on the growth of HepG2 cells,half inhibitory concentration(IC50) was (15.35±2.43)μmol/L.Sinomenine was RT inhibitor,IC50 was (21.32±2.43)μmol/L.The Western blot showed that CASP3,CASP9 and CAV1 were up-regulated and SOX2 was down-regulated by the sinomenine treatment in HepG2 cells.Conclusion: The potential molecular mechanism of sinomenine suppresses proliferation and metastasis of HepG2 cells by up-regulation of CASP3,CASP9,CAV1 and down-regulation of SOX2.

RalA regulates ROS and ATP production in cancer cells by association with cavelolin-1 / 医用生物力学

Objective By analyzing mitochondrial function, reactive oxygen species (ROS) and adenosine triphosphate (ATP) production under different levels of RalA and caveolin-1 (Cav-1) expression, to investigate the regulation role of RalA played in cancer metabolism and explore the possibility of its regulation role involved in Cav-1 and caveolae motility. Methods Firstly, RalA and Cav-1 expression were inhibited by siRNA in breast cancer cell line MDA-MB-231, and then the changes of mitochondrial membrane potential (MMP), ROS production, ATP generation and L-lactate level before and after inhibition were assessed by Western blotting, confocal microscope and fluorescence quantification. Results (1) The decreased RalA and Cav-1 expression led to a significant reduction of MMP directly. (2) Low RalA and Cav-1 expression resulted in an inhibition of ATP production and an increase of H2O2 generation. With the reduction of MMP, mitochondrial malfunction was observed. (3) With mitochondrial function suppression, an elevated level of glycolysis metabolite L-lactate was also detected in RalA and Cav-1 deprived cells. Conclusions RalA and Cav-1 mediate cellular metabolic switch by inhibiting mitochondrial function and simultaneously boosting glycolysis. This regulation role of RalA depends on its association with Cav-1, and possibly is related to the endocytosis and motility of caveolae. The research findings enrich the cancer metabolic studies, and provide a novel approach for cancer therapeutic strategy targeted to cellular metabolism.

Construction of and Protein Preparation from a Recombinant Plasmid Containing the Distal Fragment of the Distal C-terminus of the Cav1.2 Channel / 中国医科大学学报

Objective To construct a recombinant plasmid vector containing the distal fragment of the distal C-terminus (dDCT) of the Cav 1.2 channel,and express,extract,and purify dDCT protein and characterize its biological activity.Methods dDCT cDNA was ligated into the pGEX-6p-1 vector to create a recombinant plasmid that was subsequently transformed into Escherichia coli BL21 competent cells.Expression of GST-dDCT fusion protein from this plasmid was induced with isopropy-β-D-thiogalactoside,and the resulting protein was purified using glutathione-sepharose 4B beads.The biological activity of dDCT was analyzed by GST pull-down assay.Results The recombinant plasmid was verified by restriction enzyme digestion and sequencing.The concentration and purity of the dDCT protein,which was extracted by ultrasonication,were high enough to detect dDCT activity.The binding of dDCT to CT1 was determined to be concentration-dependent.Conclusion The recombinant dDCT plasmid was successfully constructed,providing the fundamental basis for future studies on mechanisms of Cav 1.2 channel autoregulation.

Evaluation of the CaV1 1-R528 H gene knock-in mouse model of thyrotoxic hypokalemic periodic paralysis / 中国实验动物学报

Objective To establish and evaluate the CaV1?1?R528H gene knock?in mouse model of thyrotoxic hy?pokalemic periodic paralysis. Methods Thirty?six 8?week?old male CaV1?1?R528H gene knock?in mice and thirty?six 8?week?old wild?type male C57BL/6J mice were used in this study. Using three?factor two?level 2 × 2 × 2 factorial design ( the three factors including mutation, thyroxine and insulin, and two levels were with or without) , the mice were divided into 8 groups. The thyroxine groups were intraperitoneally injected with levothyroxine in a dose of 350 μg/kg once per day for 12 consecutive days to produce thyrotoxicosis. The insulin groups were intraperitoneally injected with short?acting insulin in a dose of 0?8 U/kg after the last administration of levothyroxine, and the potassium levels of different groups were meas?ured and recorded before (0 min) and after insulin injection (30 min, 60 min). Results (1) Compared with the control group, the following phenomena including irritability, dull coat, increased diet and water intake, and slow body weight gain, were observed in the thyrotoxic mice. Thyroid function tests showed that the levels of T3 and T4 in the thyrotoxic mice were significantly higher than those in the corresponding control mice (P<0?05), and the TSH level was significantly low?er than that of the corresponding control mice (P<0?05 ). (2) After administration of insulin or thyroxine alone, the po?tassium levels in the mutant and wild?type mice were not significantly different. However, after combined administration of thyroxine and insulin, the potassium levels in the mutant group were significantly lower than those in the wild?type mice at 30 min and 60 min ( P<0?05 for both). (3) The main effects and interactions:Mutation factor or thyroxine factor alone did not influence on the potassium level, only insulin showed hypokalemic effect (P<0?05). There were interactions be?tween thyroxine and mutation, and between insulin and mutation (P<0?05), but no interaction between thyroxine and in?sulin. Conclusions (1) A thyrotoxicosis state in mice is successfully developed in this study. (2) An CaV1?1?R528H gene knock?in mouse model of thyrotoxic hypokalemic periodic paralysis is successfully established.

Effect of root saponins of Panax notoginseng on different voltage-dependent calcium and potassium ion channels / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of root saponins of Panax notoginseng(RPNS) on different voltage-dependent calcium and potassium ion channels. METHODS By using the two-elec?trode voltage clamp (TEVC), the effect of RPNS 0.01, 0.06, 0.1, 0.6, 1 and 4 g · L- 1 was investigated on Cav1.2,and the effect of RPNS 1 g · L-1 was evaluated on Cav2.1,Cav2.2,Cav3.1, KCNH2,KCNQ1,KCNQ1/KCNE1 and BK channel. All the ion channels examined were expressed in Xenopus laevis oocytes. RESULTS TEVC suggested that the effect of RPNS on Cav1.2 exhibited the concentration-response relationship and its EC50 was 0.048 g · L-1. Compared with cell control,TEVC also showed that RPNS 1 g·L-1 had obviously inhibitory effect on Cav1.2,Cav2.2 and Cav3.1,and the inhibitory rate of RPNS 1 g · L-1 on the peak current of Cav1.2,Cav2.2 and Cav3.1 was(57.1 ± 8.6)%, (17.2 ± 0.7)% and(50.2 ± 7.7)%(P<0.01),respectively. RPNS 1 g · L-1 had obviously activated effect on BK channel,and the activated rate of RPNS 1 g·L-1 on the peak current of BK channel was(37.9± 2.7)%(P<0.01). RPNS 1 g·L-1 showed no significant effect on Cav2.1,KCNH2,KCNQ1 and KCNQ1/KCNE1. CONCLUSION RPNS may effectively inhibit Cav1.2 and Cav3.1,activate BK channel,but have little effect on Cav2.1,Cav2.2,KCNH2,KCNQ1 and KCNQ1/KCNE1.

Abnormal expression and its regulation mechanism of Cav-1 gene in gastric cancer / 临床与实验病理学杂志

Purpose To investigate the expression of Cav-1 in gastric cancer ( GC) cell lines and GC samples, and to analyze the pos-sible effect of gene methylation in expression of Cav-1. Methods Methylation specific PCR ( MSP) method was applied to examine the CpG methylation of the Cav-1 promoter in GC cell lines (AGS, MKN45, BGC-823) and 104 samples of GC and corresponding ad-jacent tissues. RT-PCR method was applied to examine the mRNA expression in GC cell lines. IHC were applied to examine the pro-tein expression of Cav-1 in the GC tissues. Results The expression level of Cav-1 mRNA was obviously increased after treated with 5-aza-2’-deoxycytidine (5-Aza-Dc, a demethylation agent) in AGS cell line. We detected the positive expression of Cav-1 gene in MKN45 and BGC-823 cell lines before and after treated with 5-Aza-Dc. The level of Cav-1 mRNA expression was no any change in AGS, MKN45 and BGC-823 cell lines treated with trichostatin A (TSA). MSP results showed that it can be amplified methylated bands in AGS cell line, and the methylated bands disappeared after treated with 5-Aza-Dc. MKN45 and BGC-823 cell lines were no any methylated bands amplified before and after treatment. The methylation frequency of Cav-1 gene was 29. 8% (31/104), which was significantly higher than that in adjacent tissues (P=0. 000). Furthermore, Cav-1 gene hypermethylation status was correlated with lymph node metastasis and family history of upper gastrointestinal cancers ( UGIC) , but not with pathological grade and clinical stage (P>0. 05). The positive frequency of Cav-1 expression was 51. 9%(54/104) in GC, which was significantly lower than that in adja-cent tissues (P=0. 000). The expression of Cav-1 was correlated with the frequency of gene methylation in GC tissues (P=0. 000). Conclusion The expression of Cav-1 was reduced in GC tissues and the gene hyermethylation may be one of the mechanisms causing Cav-1 gene silencing.