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Objective: To establish and evaluate a microbial sensitivity test method for Neisseria gonorrhoeae based on resazurin coloration., Methods: Based on the broth microdilution method, resazurin was added as a live bacteria indicator. WHO G, a WHO gonococcal reference strain, was used to optimize the incubation time for resazurin-stained bacteria and the color change was visually observed to obtain the results. Agar dilution method (the gold standard) and resazurin-based microdilution assay were used to determine the minimum inhibitory concentration (MIC) of azithromycin, ceftriaxone, and spectinomycin for 3 reference strains and 32 isolates of Neisseria gonorrhoeae . The results were analyzed based on essential agreement (EA), which reflected the consistency of the MIC values, category agreement (CA), which reflected the consistency in the determination of drug resistance, intermediary, and sensitivity, very major error (VME), which reflected false sensitivity, and major error (ME), which reflected pseudo drug resistance, to evaluate the accuracy of resazurin-based microdilution assay as a microbial sensitivity test of of Neisseria gonorrhoeae . CA and EA rates≥90% and VME and ME rates≤3% were found to be the acceptable performance rates., Results: The results obtained 6 hours after resazurin was added were consistent with those of the agar dilution method and the resazurin-based microdilution assay was established accordingly based on this parameter. The EA of resazurin-based microdilution assay for measuring the MIC results of azithromycin, ceftriaxone, and spectinomycin was 97.1%, 91.5%, and 94.3%, respectively, and the CA was 88.6%, 94.3%, and 94.3%, respectively. The VME was 0% for all three antibiotics, while the ME was 11.4%, 5.7%, and 5.7%, respectively., Conclusion: The resazurin-based microdilution assay established in this study showed good agreement with agar dilution method for measuring the MIC of antibiotics against Neisseria gonorrhoeae . Moreover, the sensitivity results of this method were highly reliable and could be easily obtained through naked eye observation. Nonetheless, the results of drug resistance should be treated with caution and the optimization of parameters should be continued., Competing Interests: 利益冲突　所有作者均声明不存在利益冲突, (© 2024《四川大学学报（医学版）》编辑部 版权所有Copyright ©2024 Editorial Board of Journal of Sichuan University (Medical Sciences).)

Objective: To investigate the effect and related mechanism of homocysteine (Hcy) on calcium overload in neonatal rat atrial cells (NRICs). Methods: NRICs were assigned to 9 groups after culture for 3 days: (1) control group; (2) Hcy group (0, 50, 100, 200, 500 μmol/L for 48 hours); (3) antioxidant group (NAC, 10 μmol/L for 24 hours); (4) Hcy+NAC group (500 μmol/L Hcy for 48 hours, then treated with 10 μmol/L NAC for 24 hours); (5) calcium/calmodulin dependent protein kinase Ⅱδ (CaMKⅡδ) inhibitor group (KN-93, 3 μmol/L KN-93 for 5 hours); (6) specific sodium current inhibitor group (ELE, 1 μmol/L ELE for 5 hours); (7) Hcy+KN-93 group (500 μmol/L Hcy for 48 hours, then treated with 3 μmol/L KN-93 for 5 hours); (8) Hcy+ELE group (500 μmol/L Hcy for 48 hours, then treated with 1 μmol/L ELE for 5 hours; (9) Hcy+KN-93+ELE group (500 μmol/L Hcy for 48 hours, then treated with 3 μmol/L KN-93 and 1 μmol/L ELE for 5 hours). Moreover, NRICs were also treated with CaMKⅡδ-siRNA lentivirus, and Nav1.5-siRNA lentivirus, negative lentivirus carrier containing green fluorescent protein (GFP) for 24 hours. The MOI values of the three groups were 10. Infection efficiency of lentivirus was determined by observing the percentage of GFP fluorescence under inverted fluorescence microscope after transfection for 24 hours, and cultured regularly with simultaneous Puro screening, then cells were grouped as Hcy+CaMKⅡδ-siRNA group, Hcy+Nav1.5-siRNA group and Hcy+negative group. The concentration of Ca(2+) in NRICs ([Ca(2+)]i) of various groups was detected through Fluo-4/AM fluorescence probe, then 2', 7'- two chlorofluorescein diacetate (DCFH-DA) was used as a probe to detect reactive oxygen species (ROS) in NRICs by flow cytometry. The malondialdehyde (MDA) was detected by the activity of superoxide dismutase (SOD) and xanthine oxidase was detected by thiobarbituric acid colorimetry. The protein and mRNA expression level of CaMKⅡδ and Nav1.5 in NRICs were detected by Western blot and quantitative real-time PCR. Results: (1) ROS, MDA and SOD were similar between NAC group and control group, ROS and MDA were significantly increased, while SOD was significantly reduced in Hcy group in a concentration-dependent manner. (2) [Ca(2+)]i: The level of [Ca(2+)]i was (155.57+7.25), (187.43+13.07), (248.98+27.22) and (307.36+15.09) nmol/L in 50, 100, 200 and 500 μmol/L Hcy groups, which was significantly higher than that in the control group ((123.18+7.24) nmol/L, P< 0.01). In addition, the level of [Ca(2+)]i in Hcy+NAC group ((222.87+23.71)nmol/L) was significantly lower than that in Hcy 500 μmol/L group ((305.15+39.45) nmol/L, P< 0.05), while [Ca(2+)]i level was similar between NAC group and the control group. (3) The protein expression of CaMKⅡδ and Nav1.5 was significantly upregulated in Hcy groups than in the control group. The protein expression level of CaMKⅡδ-Thr287 was significantly lower in NAC group than in Hcy 500 μmol/L group ( P< 0.01), however, there was no significant difference on the protein expression levels of CaMKⅡδ-Thr287 and Nav1.5 between NAC group and control group (all P> 0.05). (4) The protein expression levels of CaMKⅡδ-Thr287 and the concentration of [Ca(2+)]i were significantly lower in Hcy+KN-93 group and Hcy+KN-93+ELE group than in Hcy 500 μmol/L group ( P< 0.05). [Ca(2+)]i concentration was significantly lower in Hcy+KN-93 group, Hcy+ELE group and KN-93+ELE+Hcy group than in Hcy 500 μmol/L group ( P< 0.05). (5) The mRNA and protein expression levels of CaMKⅡδ and Nav1.5 in each group infected with lentivirus: the GFP expression was ideal post lentivirus transfection for 24 hours (up to 90%), which was significantly lower in the CaMKⅡδ-siRNA group and Nav1.5-siRNA group than in the negative infection group (all P< 0.05), which was similar between negative infection group and control group ( P> 0.05). Moreover, the mRNA and protein expression levels of CaMKⅡδ and CaMKⅡδ-Thr287 was significantly lower in Hcy+Nav1.5-siRNA group than in Hcy+negative infection group ( P< 0.05). The protein and mRNA levels of Nav1.5 were similar between Hcy+CaMKⅡδ-siRNA group and Hcy+negative infection group ( P> 0.05). Conclusions: Hcy can induce calcium overload in NRICs by increasing oxidative stress, upregulating the sodium channel protein, and activating the late sodium current and phosphorylating CaMKⅡδ.

Objective: To explore the effect and possible mechanisms of intermittent alkaline on rat vascular smooth muscle cells (VSMCs) calcification induced by high phosphorus. Methods: VSMCs were isolated from rat thoracic aorta and cultured in vitro. The fourth generation VSMCs were randomly divided into control group, high phosphorus+ pH7.4, high phosphorus+ pH7.5, high phosphorus+ pH7.6 and high phosphorus+ pH7.7 group with random number table. The control group was cultured in DMEM with 10% fetal bovine serum. Other groups were cultured in DMEM with 10 mmol/L β-glycerophosphate and alkalized by 7.4% NaHCO(3) to adjust the pH respectively. After the intervention of 4 hours, the control group was replaced with the normal medium containing 10% fetal bovine serum, the other 4 groups were replaced with high phosphorus based on the pH value of the culture medium, and then replaced the culture medium every other day. After 4 days intervention, the mRNA and protein expression of L type calcium channel β(3) subunit(LTCC β(3)) and Runt related transcription factor 2 (Runx2) were detected by RT-PCR and Western blot. After 4 days intervention, the level of VSMC calcium ion was detected by Fluo-3/AM. After 14 days intervention, alkaline phosphatase (ALP) activity was measured by enzyme linked immunosorbent assay (ELISA) and the calcification was observed by measuring calcium content. Results: (1) Compared with control group, the gene and protein expressions of LTCC β(3) were higher in high phosphorus+ pH7.4 group (0.49±0.03 vs. 0.23±0.02 and 0.45±0.03 vs. 0.26±0.02 respectively, all P <0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.86±0.05) and protein(0.62±0.04) expressions of LTCC β(3) were higher in high phosphorus+ pH7.5 group ( P <0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.99±0.05) and protein(0.80±0.03) expressions of LTCC β(3) were higher in high phosphorus+ pH7.5 group (all P <0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.16±0.05) and protein(0.93±0.03) expressions of LTCC β(3) were higher in high phosphorus+ pH7.7 group (all P <0.05). (2) Compared with control group, calcium ion influx were higher in high phosphorus+ pH7.4 group (124.61±6.06 vs. 75.68±7.82, P <0.05). Compared with high phosphorus+ pH7.4 group, calcium ion influx was higher in high phosphorus+ pH7.5 group(210.85±9.75, P <0.05). Compared with high phosphorus+ pH7.5 group, calcium ion influx was higher in high phosphorus+ pH7.6 group(298.44±11.42, P <0.05). Compared with high phosphorus+ pH7.6 group, calcium ion influx was higher in high phosphorus+ pH7.7 group(401.13±11.41, P <0.05). (3) Compared with control group, the mRNA and protein expressions of Runx2 and ALP were higher in high phosphorus+ pH7.4 group (0.60±0.04 vs. 0.34±0.03, 0.42±0.04 vs. 0.21±0.02, 67.2±4.3 vs. 23.2±2.3 respectively, all P <0.05). Compared with high phosphorus+ pH7.4 group, the mRNA(0.76±0.05) and protein(0.68±0.03) expressions of Runx2 and ALP(102.1±5.4) were higher in high phosphorus+ pH7.5 group (all P <0.05). Compared with high phosphorus+ pH7.5 group, the mRNA(0.90±0.05) and protein(0.90±0.05) expressions of Runx2 and ALP(139.3±4.9) were higher in high phosphorus+ pH7.6 group (all P <0.05). Compared with high phosphorus+ pH7.6 group, the mRNA(1.11±0.05) and protein(1.08±0.06) expressions of Runx2 and ALP(197.0±6.7) were higher in high phosphorus+ pH7.7 group (all P <0.05). (4) Compared with control group, the calcium content were higher in high phosphorus+ pH7.4 group ((75.4±4.3)mg/g pro vs.(25.2±2.1)mg/g pro, P <0.05). Compared with high phosphorus+ pH7.4 group, the calcium content were higher in high phosphorus+ pH7.5 group ((100.8±5.7) mg/g pro, P <0.05). Compared with high phosphorus+ pH7.5 group, the calcium content were higher in high phosphorus+ pH7.6 group ((143.5±6.1) mg/g pro, P <0.05). Compared with high phosphorus+ pH7.6 group, the calcium content were higher in high phosphorus+ pH7.7 group ((205.1±8.2) mg/g pro, P <0.05). Conclusion: Intermittent alkaline stimulation can promote high phosphorus induced rat VSMCs calcification possibly through upregulating LTCC β(3) subunit gene and protein expression, increasing calcium ion influx and enhancing VSMCs phenotypic transformation.

Objective: To detect the cytotoxicity of fine particulate matter treated on human bronchial epithelial cells( BEAS-2B) and human embryonic lung fibroblasts( MRC-5) by using the method of real time cell analyze( RTCA) and Alamar Blue., Methods: Seed the BEAS-2B cells and MRC-5 cells in the 16-well plates for RTCA analyzer and commonly 96-well plates respectively. After 24 hours, the BEAS-2B cells and the MRC-5 cells were treated by the fine particulate matter respectively in different doses( 2500, 1250, 625, 312. 5, 156. 25 and 78. 125 μg / m L). Calculating the IC50 values which determined by the two methods on different times( 3, 12, 24, 36 and 48 h). Using paired t test by SPSS 16. 0 analyse the IC50 values from two methods and estimate the correlation., Results: There was correlation between the two methods, but there was no obvious difference between the two results by RTCA and Alamar Blue., Conclusion: There is certain cytotoxicity of fine particulate matter. At the same time, the RTCA real-time monitoring technology can be used on determination of cytotoxicity in vitro.

Objective: To investigate the effects of gambogenic acid on proliferation,apoptosis and invasion of human cervical carcinoma HeLa cells., Methods: HeLa cells were given different concentrations of gambogenic acid( 0. 00,0. 63,1. 25,2. 50,5. 00 and 10. 00 mg / L) for 72 h,MTT assay was used to measure the cells proliferation. Flow cytometry was used to measure cell cycle. Fluorescence microscopy and DNA ladder analysis were used to measure the apoptosis of HeLa cells. Transwell chambers was adopted to detect the cells invasion. The expression levels of mRNA and protein of BCL-2,BAX,NF-κB and E-cadherin were detected by RT-PCR and Western blot., Results: After treatment with different concetrations of gambogenic acid for 72 h, the proliferation of HeLa cells was significantly inhibited, and in a concentration dependent manner. The ability of invasion was decreased with the concentration of gambogenic acid increased, which was detected by Transwell chamber assays in vitro. RT PCR and Western blot demonstrated that gambogenic acid up-regulated the expressions of BAX and E-cadherin and down-regulated the expression of mRNA and protein of BCL-2 and NF-κB., Conclusion: Gambogenic acid can inhibite the proliferation and invasion of HeLa cells,and promote the cells apoptosis in a concentration dependent manner.

Objective: To investigate the regulation mechanism of estrogen on the free calcium of smooth muscle cells at the endometrial-myometrial interface (EMI) in uteri with adenomyosis., Methods: From September 2011 to November 2012, 59 uterine myometrial specimens were obtained from 59 cases underwent hysterectomy, including 28 adenomyosis patients as adenomyosis (ADS) group and 31 patients with cervical intraepithelial neoplasia III as control group. EMI smooth muscle cells were cultured and loaded with calcium ion fluorescent probe fluo-4/AM. After treated with trisphosphate (IP3) receptor antagonist, blocker of sarcoplasmic reticulum calcium-adenosine triphosphate (ATP), depleted agent of the ryanodine receptor-operated Ca(2+), inhibitor of L-type calcium channel, inhibitor of Na(+)-Ca(2+) exchanger, the labeled cells were stimulated with estrogen. The changes of intracellular Ca(2+) fluorescence intensity were detected by laser scanning microscopy. The changes of intracellular Ca(2+) concentration was indicated by △F[Ca(2+)](i)., Results: (1) Under normal calcium conditions, after the stimulation of estrogen, intracellular Ca(2+) fluorescence intensity in ADS group and control group both increased than those without estrogen. The △F[Ca(2+)](i) in ADS group was 384 ± 26, and in the control group △F[Ca(2+)](i) was 235 ± 20. The △F[Ca(2+)](i) in ADS group was higher than that in the control (P < 0.01). Without calcium conditions, the △F[Ca(2+)](i) in ADS group was 207 ± 17, and in the control group △F[Ca(2+)](i) was 221 ± 19. The △F[Ca(2+)](i) in ADS group was almost the same with the increase in the control (P = 0.731). The △F[Ca(2+)](i) in ADS group was significantly decreased compared with the calcium condition (P < 0.01). However, there was no significant difference in the control between with and without calcium conditions (P = 0.060). (2) After treated with IP3 receptor antagonist, blocker of sarcoplasmic reticulum calcium-ATP, depleted agent of the ryanodine receptor-operated Ca(2+), the △F[Ca(2+)]i in both groups were significantly reduced (P < 0.05), the increase in ADS group was significantly higher than that in the control (P < 0.05). (3)After treated with inhibitor of L-type calcium channel, the △F[Ca(2+)](i) in ADS group was 211 ± 19, while in the control group △F[Ca(2+)](i) was 203 ± 16, and there was no significantly increased intracellular Ca(2+) in both groups (P > 0.05). But, the △F[Ca(2+)](i) in ADS group was significantly reduced after treatment compared to before treatment, (211 ± 19 vs 384 ± 28; P = 0.001). The increase in control group was almost the same with before (203 ± 16 vs 234 ± 22, P = 0.141). (4) After treated with inhibitor of Na(+)-Ca(2+) exchanger, the △F[Ca(2+)](i) in ADS group was 357 ± 24 and in the control △F[Ca(2+)](i)was 209 ± 19. The increase in ADS group was significant higher than that in the control (P = 0.000). Compared with △F[Ca(2+)](i) on the condition without treating with inhibitor of Na(+)-Ca(2+) exchanger, △F[Ca(2+)](i) was 363 ± 21 in ADS group and △F[Ca(2+)](i) was 237 ± 20 in control group after treatment. When compared with before treatment, there was no significant difference in both groups (P > 0.05)., Conclusions: The increase of intracellular Ca(2+) induced by estrogen at EMI smooth muscle cells in adenomyosis patients was mostly from the release of arcoplasmic reticulum, and also from the Ca(2+) influx controlled by L-type calcium channel. The increase of Ca(2+) inducing abnormal contraction of EMI muscle may have relationship with the development of adenomyosis.

Objective: To study whether the analgesis of oxymatrine (OMT) affects N-type voltage-gated calcium channels (VGCCs)., Methods: Totally 45 mice were randomly divided into the sham-operation group, the model group [established by partial sciatic nerve ligation (PSNL)] , and the OMT treatment group according to random digit table, 15 in each group. The dorsal root ganglions (DRG) were separated in PSNL pain model mice. Intracellular calcium concentration ([Ca2+]i) was determined with Fluo-3 AM immunofluorescent probe in cultured DRG neurons. Different protein expression levels of N-type (Cav2. 2) and L-type ( Cav1. 3) among VGCCs from brain and DRG tissues were detected with Western blot., Results: Compared with the sham-operation group, [Ca2+]i, increased in cultured DRG neurons (P <0. 05) , protein expression levels of Cav2. 2 in the brain tissue increased (P <0. 05), protein expression levels of Cav2. 2 in DRG tissues decreased in the model group (P <0. 01). Compared with the model group, [Ca2+]i, decreased in cultured DRG neurons (P < 0. 05), protein expression levels of Cav2. 2 in the brain tissue decreased (P <0. 01), protein expression levels of Cav2. 2 in DRG tissues increased in the OMT treatment group (P <0. 01). There was no statistical difference in Cav1. 3 expressions in cultured DRG neurons and the brain (P >0. 05)., Conclusion: Analgesic effect of OMT might be related to Cav2. 2 channel mediated calcium ion flux.

Objective: To observe the effect of κ-opioid receptor (κ-OR) stimulation on Angiotensin II (Ang II)-induced cardiomyocyte hypertrophy in vitro cultured myocardial cells from neonatal rats and on calcineurin (CaN) signal pathways., Methods: Cultured myocardial cells of neonatal rats were divided into control group, CSA (1 µmol/L) group, Ang II (1 µmol/L) group, Ang II (1 µmol/L) + U50488H (1 µmol/L) group, Ang II (1 µmol/L) + CSA (1 µmol/L) group, Ang II (1 µmol/L) + Rp-cAMPS (1 µmol/L) group, Ang II (1 µmol/L) + CSA (1 µmol/L) + U50488H (1 µmol/L) group and Ang II (1 µmol/L) + PTX5 mg/L + U50488H (1 µmol/L) group. The hypertrophic myocytes were induced by Ang II 1µmol/L before κ-OR agonist U50488H 1 µmol/L was administered. The antihypertrophic effect of κ-OR stimulation was observed in the presence of ciclosporine A (CsA) 1 µmol/L, cAMP triethyl-ammonium salt (Rp-cAMPS) 1 µmol/L, and pertussistoxin ( PTX) 5 mg/L. The total protein content was assayed by the method of Lowry. The [Ca²⁺]i was measured by confocal microscope using Fluo-3/AM as flouresecent indicator. The relative expression of CaN was determined by Western blot., Results: (1) The total protein content of Ang II group was significantly higher than that in control group (P<0.01), which could be equally reduced by cotreatment with U50488H, CSA and Rp-cAMPS (P<0.01). Total protein content of the Ang II + PTX + U50488H group and the Ang II group was similar. (2) The [Ca²⁺]i was significantly higher in Ang II group of neonatal rat cardiomyocytes than that in control group (P<0.01), which could be reduced by cotreatment with U50488H, CSA and Rp-cAMPS (P<0.01). [Ca(2+)]i was similar between the Ang II + PTX + U50488H group and the Ang II group. (3) The expression of CaN was significantly higher in Ang II group than that in control group (P<0.01), which could be significantly reduced by cotreatment with U50488H, CSA and Rp-cAMPS (P<0.01). CaN was similar between the Ang II + PTX + U50488H group and the Ang II group., Conclusion: κ-opioid receptor activation could attenuate Ang II induced cardiomyocytes hypertrophy via reducing [Ca²⁺]i and downreglating CaN.

To investigate the neuroprotective of ligustilide (LIG) against glutamate-induced apoptosis of PC12 cells, cell viability were examined by MTT assay. Flow cytometry was applied to assay cell apoptosis rate. Intracellular calcium concentration was measured by using fluorescent dye Fluo-3/AM. Cytochrome C (Cyt C), Caspase-3, Bax and Bcl-2 protein expression were assayed by western blot. The results showed that glutamate is cytotoxic with an inhibitory concentration 50 (ID50) of 15 mmol · L(-1). Pretreatment with LIG (1, 5, 15 μmol · L(-1)) significantly improved cell viability. The apoptosis rate in glutamate-induced PC12 cells was 13.39%, and decreased in the presence of LIG (1, 5, 15 μmol · L(-1)) by 9.06%, 6.48%, 3.82%, separately. Extracellular accumulation of Ca2+ induced by glutamate were significantly reduced by LIG. The results of western blot manifested that pretreatment LIG could decrease the release of Cyt C from mitochondria, down-regulate Caspase-3 protein expression and up-regulate Bcl-2/Bax ratio, thereby protects PC12 cells from apoptosis. In summary, LIG had protective effect on glutamate-induced apoptosis in PC12 cells through attenuating the increase in intracellular Ca2+ concentration, and inhibiting the release of Cyt C from mitochondria to cytoplasm.

Objective: To study the effects of mitochondrial permeability transition (MPT) on hepatocyte apoptosis and mitochondrial damage, and investigate the effects of curcumin on MPT and the related mechanisms in septic rat., Methods: Fifteen healthy male Sprague-Dawley (SD) rats were randomly divided into three groups: sham group, sepsis group and curcumin group, with 5 rats in each group. Septic rat model was reproduced by cecal ligation and puncture (CLP). The rats in sham group were flipped the cecum without perforation and ligation. The rats in the curcumin group were treated with curcumin 100 mg × kg⁻¹ × d⁻¹ (dissolved in saline to 10 mL/kg) by oral gavage for 7 days, while the other groups were treated with normal saline. Tissue samples were harvested in each group at 12 hours after operation. Pathological changes in hepatic mitochondria were observed under electron microscopy, concentration of free calcium was examined with confocal laser scanning microscope. After Fluo-3/AM staining, protein and mRNA expression of active caspase-3, Bcl-2 and Bax were examined by Western Blot and reverse transcription-polymerase chain reaction (RT-PCR)., Results: Under the transmission electron microscope, intact cell membrane, adqulis cytoplasm, and normal and clear mitochondrion was found in the sham group. Mitochondria in sepsis group swelled obviously with mitochondrial cristae broken or disappearance, unclear bilateral membrane structure, while the curcumin group showed much less pathological changes, with few mitochondria swell, and smear bilateral membrane structure. The fluorescence intensity index of sham group, sepsis group and curcumin group was raised successively (417.33 ± 15.88, 772.95 ± 42.37, 1 560.84 ± 160.78, respectively, F=184.149, P=0.000). The protein and mRNA expression of active caspase-3 and Bax had the highest level in sepsis group, followed by the curcumin group, and that in the sham group was the lowest [active caspase-3 protein (gray scale): 1.698 ± 0.061, 0.694 ± 0.045, 0.246 ± 0.027, F=1 289.667, P=0.000; active caspase-3 mRNA (2(-ΔΔCt)): 1.031 ± 0.135, 0.578 ± 0.144, 0.183 ± 0.036, F=66.958, P=0.000; Bax protein (gray scale): 1.826 ± 0.126, 1.254 ± 0.140, 0.623 ± 0.901, F=94.536, P=0.000; Bax mRNA (2(-ΔΔCt)): 2.774 ± 0.338, 1.661 ± 0.226, 0.656 ± 0.114, F=124.710, P=0.000], all of these values had statistical significance among the three groups (all P<0.01). While Bcl-2 protein and mRNA had the highest level in curcumin group and lowest level in the sham group [Bcl-2 protein (gray scale): 0.716 ± 0.091, 1.328 ± 0.147, 1.656 ± 0.104, F=84.918, P=0.000; Bcl-2 mRNA (2(-ΔΔCt)): 0.617 ± 0.118, 1.393 ± 0.096, 1.650 ± 0.167, F=83.846, P=0.000]. The protein and mRNA expressions of Bcl-2/Bax ratio were lowest in sepsis group, then sham group, and highest in curcumin group [Bcl-2/Bax protein (gray scale): 0.726 ± 0.055, 1.150 ± 0.043, 1.333 ± 0.163, F=46.265, P=0.000; Bcl-2/Bax mRNA (2(-ΔΔCt)): 0.505 ± 0.041, 0.944 ± 0.097, 1.006 ± 0.168, F=12.211, P=0.001]., Conclusions: MPT can lead to mitochondrial dysfunction and further cause hepatocyte apoptosis. Mechanism of effect of curcumin on MPT may be related to reduction of intracellular calcium concentration, promotion of anti-apoptotic Bcl-2 gene expression, inhibition of caspase-3 activation and Bax gene.

Objective: To discuss the mechanism of gambogenic acid (GNA) in inducing the apoptosis of melanoma B16 cells., Method: The inhibitory effect of GNA on the proliferation of B16 cells was measured by the methyl thiazolyl tetrazolium (MTT) assay. The effect of GNA on B16 cells was detected by the Hoechst 33258 staining. The transmission electron microscopy was used to observe the ultra-structure changes of B16 cells. The changes in PI3K, p-PI3K, Akt, p-Akt, p-mTOR, PTEN proteins were detected by the Western blotting to discuss the molecular mechanism of GNA in inducing the apoptosis of B16 cells., Result: GNA showed a significant inhibitory effect in the growth and proliferation of melanoma B16 cells. The cell viability remarkably decreased with the increase of GNA concentration and the extension of the action time. The results of the Hoechst 33258 staining showed that cells processed with GNA demonstrated apparent apoptotic characteristics. Under the transmission electron microscope, B16 cells, after being treated with GNA, showed obvious morphological changes of apoptosis. The Western blot showed a time-dependent reduction in the p-PI3K and p-Akt protein expressions, with no change in p-PI3K and p-Akt protein expression quantities. The p-mTOR protein expression decreased with the extension of time, where as the PTEN protein expression showed a time-dependent increase., Conclusion: GNA could inhibit the proliferation of melanoma B16 cells and induce their apoptosis within certain time and concentration ranges. Its mechanism in inducing the cell apoptosis may be related to PI3K/Akt/mTOR signaling pathways.

To explore gambogenic acid (GNA)-induced apoptosis and underlying mechanism in vivo. A549 nude mice xenografts were used as in vivo model to study anticancer effect of GNA by observing tumor growth curve and weight of the tumor. Ultrastructure of A549 cells treated by GNA was observed by TE. Expression of COX-2 and VEGF were detected by immunohistochemistry. TUNEL assay was applied in examining apoptosis index of tumor cells. The tumor isolated from mice treated by GNA (8, 16 mg kg(-1)) took on a slow growth condition compared with control group. The results suggested that weight and volume of the tumor from experimental groups were remarkably decreased compared with control group (P < 0.05). Ultrastructure change of the tumor, such as vacuolization, abnormal distribution of the heterochromosome, volume of the tumor cells, even apoptotic bodies, were observed in GNA-treated group. While no apparent morphological change was observed in the normal group. Typical apoptotic characteristics could be distinctly observed in the mouse treated by GNA for 20 days and apoptosis index in GNA-treated group was significantly higher than model group. Expression of COX-2 and VEGF were significantly down-regulated in GNA-treated groups in comparison with control group (P < 0.01). These results indicate that GNA could affect the development and progression of A549 cells through inducing apoptosis, mediating the expression of VEGF in vascular cells and COX-2 in tumor cells.

Objective: To observe the effects of 17beta-estradiol on the intracellular calcium of masticatory muscles myoblast., Methods: Myoblasts from maxillofacial skeletal muscle of one week old female Sprague-Dawley rats were cultured. Fluo-4-AM as the Ca2+ indicator and the laser confocal microscope system were used to observe the effects of estrogen on the cytoplasmic Ca2+ concentration in the normal pH condition and the acid condition (pH = 6.7)., Results: In the normal pH condition, when 17beta-estradiol (10(-9), 10(-8), 10(-7) mol/L) were added to cells cytoplasmic Ca2+ immediately increased then decreased right away, and in the end came into a new Ca2+ homeostasis in the base line. In the acid condition, 17beta-estradiol (10(-9), 10(-8), 10(-7) mol/L) made the cytoplasmic Ca2+ decreased immediately then came into a new Ca2+ homeostasis under the base line., Conclusion: The results suggest that estrogen may maintain the skeletal cytoplasmic Ca2+ concentration in a lower level and reduce the cytoplasmic Ca2+ accumulation to keep the normal functions of masticatory muscles myoblast.

Objective: To study the change of Ca2+ density in cultured osteoclast-like cells in response to fluid shear stress., Methods: Laser scanning confocal microscope and fluorescent probe were used to detect the free Ca2+ in osteoclast-like cells before and after undergoing fluid shear stress. The images were analyzed and compared with image software., Results: At 37 degrees C the free Ca2+ in osteoclast-like cells could be labelled effectively with 10 micromol/L Fluo-3/AM. Compared with contol group, the average intensity of Ca2+ fluorescent signal in osteoclast-like cells undergoing fluid shear stress increased significantly., Conclusion: The Cal2+ concentration in bone-marrow derived osteoclast-like cells is sensitive to fluid shear stress, which suggests osteoclast-like cells modulate their function in response to fluid shear stress through the change of free Ca2+ concentration.

Objective: To study the spatial distribution of free Ca2+ in osteoclast-like cells cultured on glass., Methods: To detect the free Ca2+ in osteoclast-like cells, the images were analyzed with image software, using the laser scanning confocal microscope and fluorescent probe., Results: At 37 degrees C the free Ca2+ in osteoclast-like cells could be labelled effectively with 10 micromol/L Fluo-3/AM, the intensity of Ca2+ fluorescent signal in the central part was greater than that in the peripheral part and in the same section the signal was not distributed evenly., Conclusion: The intensity of Ca2+ fluorescent signal is different among various organellae in osteoclast-like cell, which suggests the osteoclast-like cell modulate its own function through the spatial difference of free Ca2+ concentration.

Objective: To investigate the effects of advanced glycation end-products (AGEs) on transient cytosolic free calcium in neonatal rat cardiac myocytes (CMs) cultured in vitro., Methods: CMs cultured for 3 to 5 days in vitro were incubated with Ca(2+)-sensitive fluorescent indicator Fluo-3AM with light screening at 37 degrees celsius; with 5% CO(2) for 60 min. Changes of the fluorescence signal of free calcium caused by AGEs were measured under laser scanning confocal microscope (LSCM)., Results: Compared with the control cells, AGEs caused an increase in the concentration of cytosolic free calcium in a dose-dependent manner., Conclusion: AGEs may impair neonatal rat CMs by altering cytosolic free calcium concentration.

Objective: To investigate the effect of intracellular-free Ca2+ concentration ([Ca2+]i) on injury following transient Mg(2+)-free treatment in vitro in developing cortical neurons., Methods: Embryo cortical neurons of rats cultured for 6 d and 17 d were directly exposed to Mg(2+)-free media, or pretreated with NMDA receptor antagonists or calcium channel antagonist before being exposed to Mg(2+)-free media. MTT assay was used to study the injury of neurons. [Ca2+]i were measured using fluo-3, a fluorescent calcium-sensitive dye and laser-scanning confocal microscope, and calculated by the fluorescent intensity., Results: Compared with control, MTT conversion rates decreased after transient (3 h) Mg(2+)-free treatment in neurons cultured for 6 d and 17 d in vitro, (59.1 +/- 6.87)% and (51.2 +/- 5.90)%, respectively. In neurons pre- and co-treated with 10 mumol.L-1 MK-801, 50 mumol.L-1 AP-5 and 10 mumol.L-1 nifedipine, MTT conversion rates were higher than those of neurons with only Mg(2+)-free treatment. Peak values of [Ca2+]i in neurons cultured for 6 d and 17 d were 2.4 +/- 0.23 and 3.2 +/- 0.32, respectively. Peak value of neurons 17 d in vitro was significantly higher than that of neurons 6 d in vitro (P < 0.05). In neurons pre- and co-treated with MK-801, AP-5 and nifedipine, [Ca2+]i were lower than those of neurons with only Mg(2+)-free treatment., Conclusion: Neuronal injury and [Ca2+]i changes following Mg(2+)-free-treatment-induced seizure were different between neurons 6 d and 17 d in vitro. It suggested that the age-dependent [Ca2+]i changes might play a role in an age-dependent manner of injury following Mg(2+)-free-treatment-induced seizure. NMDA receptor-Ca2+ pathway activation was crucial in the [Ca2+]i change and the cellular injury induced by Mg(2+)-free treatment.

Objective: To study the relationship between the calcium signal and activation of mast cells, laying the foundation for discovering a new potential theraputical target of signal transduction for type I anaphylactic diseases like asthma., Methods: The fluorescent Ca2+ indicator Fluo-3 was used to observe and quantitate the calcium signal directly in activated RBL-2H3 mast cells by a flow cytometer and a confocal fluorescence microscope with laser., Results: Antigens elicited significant increase in intracellular Ca2+ concentration ([Ca2+]i) and histamine release in sensitized cells, maximal [Ca2+]i increase being reached (525 +/- 42) nmol/L (n = 12) within 90-110 s after stimulation. Increase of fluo-3-fluorescence intensity was also observed with the confocal fluorescence microscope in cytosol. No significant increase of [Ca2+]i was observed after addition of EDTA in sensitized cells from an initial value of (79 +/- 3) nmol/L, to (80 +/- 4) nmol/L (P > 0.05, n = 12), but tyrosine phosphorylation was readily observed, and the indirect fluorescent intensity was significantly greater than that in the control group. No significant change of [Ca2+]i was observed with the addition of DNP-BSA after PMA (P > 0.05, n = 8)., Conclusion: Calcium is involved in the activation process of mast cells stimulated by antigens.

Calcium distribution and mobilization during mechanical stimulation in outer hair cells of the guinea pig were monitored using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes. Spatial calcium gradients were revealed among various subcellular areas. The ratios of the fluorescence intensity of fluo-3 and fura-red were 1.71 +/- 0.85, 1.61 +/- 0.75, 1.47 +/- 0.65 and 1.39 +/- 0.66 for the cytoplasm, the cytoplasmic membrane, the cuticular plate and the nucleus respectively, indicating that free calcium ion concentrations are the highest in the cytoplasm and the lowest in the nucleus. While the calcium concentration remained relatively constant under resting conditions, it increased during mechanical stimulation. The results show that confocal ratio imaging of fluo-3 and fura-red enables us to determine more accurately the subcellular calcium distribution and that the calcium ions make a contribution to the mechanic-electrical transduction in hair cells.

It is well known that calcium is the key second intracellular messenger. Techniques using calcium fluorescent probes have been rapidly developed during the last decade, and have become one of the most important methods to measure intracellular free Ca2+ concentrations. This trend was even more accelerated after the development of a new generation of calcium fluorescent probes and the laser scanning confocal microscopy. In this article we will review the development of calcium fluorescent probes, the mechanisms and methods of measuring intracellular free Ca2+ concentrations, the properties of new calcium fluorescent probes, and their applications in life sciences.

Five natural products were isolated from S. tetraptera var. xinglongensis of which four were proved to be oleanolic acid, 1,3-dihydroxy-4,7-dimethoxyxanthone, 1-hydroxy-2,3,5-trimethoxyxanthone and beta-sitosterol. 1,3-dihydroxy-4,7-dimethoxyxanthone has been obtained for the first time from the genus Swertia.