Effects of pramipexole combined with levodopa on cognitive and mitochondrial function of rats after global cerebral ischemia-reperfusion injury / 中国康复理论与实践

ObjectiveTo investigate the effects of dopamine receptor agonist pramipexole and levodopa on emotion and cognition, and mitochondrial membrane potential of rats after global cerebral ischemia-reperfusion injury. MethodsA total of 80 male Sprague-Dawley rats were divided into sham group (n = 20), model group (n = 20), pramipexole group (n = 20) and combined group (n = 20). The latter three groups were used to prepare the model of global cerebral ischemia-reperfusion injury with Pulsinelli's four-vessel occlusion. The pramipexole group was intraperitoneally injected pramipexole 0.5 mg/kg once a day, while the combined group was injected levodopa 50 mg/kg and pramipexole 0.5 mg/kg, for 14 days. Five rats in each group were tested with open field test three, seven and 14 days after modeling; five were tested with Y-maze test seven and 14 days after modeling; five were detected mitochondrial membrane potential three, seven and 14 days after modeling; and five were observed under Nissl's staining14 days after modeling. ResultsCompared with the model group, the number of entries into the central zone (P < 0.05), total distance travelled (P < 0.05) and average velocity (P < 0.05) in the open field test increased in the pramipexole and combined groups seven and 14 days after modeling, duration spent in the central zone increased in the pramipexole and combined groups seven days after modeling (P < 0.05); the rate of spontaneous alternation of Y-maze test increased in the pramipexole and combined groups 14 days after modeling (P < 0.05); mitochondrial membrane potential in hippocampus increased in the pramipexole and combined groups seven and 14 days after modeling (P < 0.05), and it was less in the pramipexole group than in the combined group 14 days after modeling (P < 0.05); and the number of surviving neurons in the hippocampal CA1 increased in the pramipexole and combined groups 14 days after modeling (P < 0.05). ConclusionPramipexole may improve emotion and cognition of rats after global cerebral ischemia-reperfusion injury, and it may be helpful for restoring mitochondrial membrane potential as combining with levodopa.

Cytotoxic Activity of Ethanolic Extract Aquilaria malaccensis Leaves Against MCF-7 Cells

@#Introduction: Aquilaria malaccensis, also known as “Pokok Karas” in Malaysia, is widely used in Southeast Asian countries for the treatment of joint pain, diarrhoea and inflammatory diseases, and has shown beneficial effects as an anticancer agent. The aim of this study was to investigate the effect of ethanol leaf extracts of A. malaccensis on MCF-7 cells. Methods: MTT-based cytotoxic and antiproliferative assay was used to determine the outcome of ethanolic extract toward MCF-7 cells. The mode of cell death was determined by the AO/PI double staining assay and the depolarisation of the mitochondria membrane potential. Results: IC50 value of the extract against MCF-7 cells treated for 72 hours was 4.1 ± 2.08 µg/mL, while the IC50 value for doxorubicin was 2.92 ± 0.12 µg/mL. The extract showed a lower cytotoxic effect against the NIH/3T3 cells and inhibited the growth of MCF-7 cells in a dose dependent manner. AO/PI double stain showed that the ethanolic extract of A. malaccensis leaves induced MCF-7 cells into apoptotic cell death. The present study showed that the ethanolic extract of A. malaccensis induced apoptosis through mitochondrial pathway as indicated by its ability to take up JC-1. Conclusion: The study found that ethanolic extract obtained from A. malaccensis leaves is cytotoxic on MCF-7 cells, resulting to apoptotic cell death of the cells.

Mitochondrial protection effect of sirtuin2 inhibitor AGK2 on thioacetamide-induced L02 cells / 中国医师杂志

Objective:To explore the protective effect of AGK2, a selective inhibitor of sirtuin 2 (SIRT2), on the mitochondria of L02 hepatocytes induced by thioacetamide (TAA) and its related mechanism.Methods:Human-derived hepatocyte line L02 cells were cultured in vitro. Different concentrations of SIRT2 inhibitor AGK2 were used as intervention drugs. Cell counting kit-8 (CCK8) was used to detect the effects of different concentrations of AGK2 on the activity of L02 cells, and the appropriate concentration was selected as the AGK2 intervention group. The normal group was not given any drug intervention. The model group was given 90 mmol/L TAA for modeling. Low, medium and high dose AGK2 groups were added with 1, 2 and 4 μmol/L AGK2, respectively 2 h before modeling. CCK8 was used to detect cell activity in each group. Morphological changes of cells were observed under inverted light microscope. The relative protein expression levels of isocitrate dehydrogenase (IDH1), malate dehydrogenase (MDH1), SIRT2 and fission protein 1 homologue (FIS1) were detected by Western blot. The expression of SIRT2 in cells of each group was observed by confocal laser scanning microscope. The mitochondrial membrane potential of cells in each group was observed under a fluorescence microscope. Results:When AGK2 concentration was 1, 2 and 4 μmol/L, the survival rate of cells were 98.05%, 95.76% and 91.65%, respectively, with no statistical significance compared with normal group (all P>0.05). When AGK2 concentration was 8, 16, 32, 64, 128 μmol/L, the cell survival rate was significantly decreased compared with normal group (all P<0.05). Compared with the model group, the L02 cells in low, medium and high AGK2 groups had better activity and adherence, and the floating cells were significantly reduced. The higher the concentration of AGK2, the better the cell activity and adherence, and the less floating cells. Compared with the model group, the red fluorescence of L02 cells in AGK2 group was enhanced, while the green fluorescence was weakened. The higher the AGK2 concentration was, the stronger the red fluorescence was, and the weaker the green fluorescence was. Compared with the model group, the fluorescence of SIRT2 in L02 cells of low, medium and high AGK2 groups was weakened, and the higher the concentration of AGK2, the weaker the fluorescence of SIRT2. The protein expressions of IDH1 and MDH1 in L02 cells of low, medium and high AGK2 groups were significantly higher than those of model group (all P<0.05), and were positively correlated with the concentration of AGK2 ( r=0.818, P<0.05; r=0.960, P<0.05); the protein expressions of SIRT2 and FIS1 were significantly lower than those of the model group (all P<0.05), and were negatively correlated with the concentration of AGK2 ( r=-0.992, P<0.05; r=-0.998, P<0.05). Conclusions:AGK2 can reduce the mitochondrial membrane potential stimulated by TAA in L02 cells, increase the protein expression of IDH1 and MDH1, and inhibit the protein expression of SIRT2 and FIS1 in L02 cells in a dose-dependent manner.

Mitochondria targeting drugs for neurodegenerative diseases-Design, mechanism and application

Neurodegenerative diseases (NDDs) such as Alzheimer's disease (AD) and Parkinson's disease (PD) are a heterogeneous group of disorders characterized by progressive degeneration of neurons. NDDs threaten the lives of millions of people worldwide and regretfully remain incurable. It is well accepted that dysfunction of mitochondria underlies the pathogenesis of NDDs. Dysfunction of mitochondria results in energy depletion, oxidative stress, calcium overloading, caspases activation, which dominates the neuronal death of NDDs. Therefore, mitochondria are the preferred target for intervention of NDDs. So far various mitochondria-targeting drugs have been developed and delightfully some of them demonstrate promising outcome, though there are still some obstacles such as targeting specificity, delivery capacity hindering the drugs development. In present review, we will elaborately address 1) the strategy to design mitochondria targeting drugs, 2) the rescue mechanism of respective mitochondria targeting drugs, 3) how to evaluate the therapeutic effect. Hopefully this review will provide comprehensive knowledge for understanding how to develop more effective drugs for the treatment of NDDs.

Effect of Curcumin on Apoptosis of Acute T-Lymphoblastic Leukemia Cells induced by UMI-77 and Its Related Mechanism / 中国实验血液学杂志

AbstractObjective: To explore the effect and mechanism of curcumin on human T-cell acute lymphoblastic leukemia (T-ALL) cell apoptosis induced by Mcl-1 small molecule inhibitors UMI-77.@*METHODS@#T-ALL cell line Molt-4 was cultured, and the cells were treated with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77 for 24 h. The MTT method was used to detect the cell survival rate after different treatment; According to the results of curcumin and UMI-77, the experimental settings were divided into control group, curcumin group (20 μmol/L curcumin treated cells), UMI-77 group (15 μmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells) and curcumin+ UMI-77 group (20 μmol/L curcumin and 15 μmol/L Mcl-1 small molecule inhibitor UMI-77 treated cells), MTT method was used to detect cell proliferation inhibition rate, Annexin V-FITC/PI double staining method and TUNEL staining were used to detect cell apoptosis, DCFH-DA probe was used to detect cell reactive oxygen species, JC-1 fluorescent probe was used to detect mitochondrial membrane potential, Western blot was used to detect the expression levels of apoptosis-related proteins and Notch1 signaling pathway-related proteins.@*RESULTS@#After the treatment of Molt-4 cells with different concentrations of curcumin and Mcl-1 small molecule inhibitor UMI-77, the cell survival rate was decreased (P<0.05); Compared with the control group, the cell proliferation inhibition rate of the curcumin group and the UMI-77 group were increased, the apoptosis rate of cell was increased, the level of ROS was increased, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, and the protein expression of Bcl-2 was reduced (P<0.05); Compared with the curcumin group or UMI-77 group, the cell proliferation inhibition rate and apoptosis rate of the curcumin+UMI-77 group were further increased, and the level of ROS was increased. At the same time, the protein expression of Bax, Caspase-3 and Caspase-9 in the cells were all increased, the protein expression of Bcl-2 was reduced (P<0.05); In addition, the mitochondrial membrane potential of the cells after curcumin treatment was decreased, and the proteins expression of Notch1 and HES1 were reduced (P<0.05).@*CONCLUSION@#Curcumin can enhance the apoptosis of T-ALL cells induced by Mcl-1 small molecule inhibitor UMI-77 by reducing the mitochondrial membrane potential, the mechanism may be related to the inhibition of Notch1 signaling pathway.

Study on the mechanism of Wuzi-Yanzong-Wan-medicated serum interfering with the mitochondrial permeability transition pore in the GC-2 cell induced by atractyloside / 中国天然药物

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.

Mechanism of inhibiting proliferation of tumor cells by a new tetrahydroisoquinoline compound SYT-1 / 药学学报

In this study, we investigated the inhibitory effect of SYT-1, a new compound of tetrahydroisoquino-line, on tumor cell proliferation and underlying mechanisms. Cell counting kit-8 (CCK-8) method was used to detect cell proliferation; clone formation experiment was used to detect cell clone formation ability; JC-1 probe was used to detect cell mitochondrial membrane potential; 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) probe was used to detect intracellular reactive oxygen species; Annexin V-FITC/PI (fluorescein isothiocyanate/propidium) counterstaining method was used to detect apoptosis; Western blot assay was used to detect the expression level of related proteins. The experimental results show that SYT-1 has a significant inhibitory effect on the proliferation of six human-derived cancer cells. Among them, the inhibitory effect on breast cancer MCF-7 cells is the strongest, the half maximal inhibitory concentration (IC50) of SYT-1 of 48 h administration on MCF-7 cells is 5.87 μmol·L-1, which is better than that of cisplatin (8.92 μmol·L-1). Further studies have shown that SYT-1 can dose-dependently inhibit the monoclonal formation ability of MCF-7 cells, and can cause the mitochondrial membrane potential of the cells to decrease and the level of reactive oxygen species to increase. In addition, SYT-1 can significantly inhibit the activation of PI3K-Akt (phosphatidylinositol 3-kinase/protein kinase B) signaling pathway and induce apoptosis of MCF-7 cells. The above research results show that, as a new type of tetrahydroisoquinoline compound, SYT-1 has the potential to inhibit tumor cell proliferation.

Effects of bisphenol A on apoptosis of ovarian preantral follicular granulosa cells and ovarian development in mice / 南方医科大学学报

OBJECTIVE@#To investigate the effect of environmental estrogen bisphenol A (BPA) exposure on apoptosis of mouse ovarian preantral follicular granulosa cells and ovarian development and explore the underlying mechanism.@*METHODS@#Mouse ovarian preantral follicular granulosa cells were isolated from female ICR mice at postnatal day (PND) 10 and cultured @*RESULTS@#Compared with the control cells group, the isolated cells exposed to a low concentration of BPA (50 μmol/L) showed a significantly lowered apoptosis rate, increased mitochondrial membrane potential, and enhanced cellular proliferation (@*CONCLUSIONS@#BPA can concentration-dependently regulate the function of ovarian preantral follicular granulosa cells in mice and potentially affects both the pregnant mice and the offspring female mice in light of early ovarian development.

Protective effect of transient receptor potential melastatin 2 inhibitor A10 on oxygen glucose deprivation/reperfusion model / 浙江大学学报·医学版

:To investigate the effect of transient receptor potential melastatin 2 （TRPM2） inhibitor A10 on oxygen glucose deprivation/reperfusion （OGD/R） injury in SH-SY5Y cells．:Human neuroblastoma SH-SY5Y cells were subject to OGD/R injury，and then were divided into blank control group，model control group and A10 group randomly. The cell survival rate was detected by cell counting kit 8 （CCK-8）; the level of cellular reactive oxygen species （ROS） was detected by reactive oxygen detection kit; the mitochondrial membrane potential was detected by tetramethylrhodamine （TMRM） method; the number of apoptotic cells was detected by TUNEL apoptosis assay kit; the protein expression level of cleaved caspase 3 was detected by Western blot．:Compared with 3，20，30，50， has lower cytotoxicity and better inhibition effect on channel activity. Compared with the model control group，ROS level was reduced，the mitochondrial membrane potential was improved，the number of apoptosis cells was reduced ，and the expression of cleaved caspase 3 was significantly reduced in the A10 group（all <0.05）. : A10 can alleviate cell damage after OGD/R by inhibiting TRPM2 channel function，reducing extracellular calcium influx，reducing cell ROS levels，stabilizing mitochondrial membrane potential levels，and reducing apoptosis.

Effect of Licochalcone A on Apoptosis in Human Breast Cancer MDA-MB-231 Cells / 中国实验方剂学杂志

Objective:To investigate the effect of licochalcone A （LCA） on apoptosis in human breast cancer MDA-MB-231 cells， and to explore its possible mechanism. Method:MDA-MB-231 cells were treated with LCA of different concentrations， and<italic> </italic>cell counting kit-8 （CCK-8） assay was used to detect the cell viability. The cells were treated with LCA （10， 20， and 40 μmol·L^-1） for 24 h， and apoptosis was detected by Annexin V staining with fluorescein isothiocyanate （FITC） and propidium iodide （PI） （Annexin V-FITC/PI）. The level of intracellular reactive oxygen species （ROS） was detected by 2′，7′-dichlorodihydrofluorescein diacetate （DCFA-DA） fluorescent probe. Mitochondrial membrane potential （MMP） was detected by 5， 5′， 6， 6′-tetrachloro-1， 1′， 3， 3′-tetraethyl-imidacarbocyanine （JC-1） fluorescence probe. Western blot was used to detect the expression of cell apoptosis-related proteins， such as B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax）， and endoplasmic reticulum （ER） stress-related proteins， such as C/EBP homologous protein （CHOP）， activating transcription factor 4 （ATF4）， protein kinase R-like ER kinase （PERK）， p-PERK， eukaryotic translation initiation factor 2 alpha （eIF2<italic>α</italic>）， and p-eIF2<italic>α</italic>. Result:With the increase in the drug concentration （starting from 5 μmol·L^-1）， the cell viability decreased （<italic>P<</italic>0.05） with IC₅₀of 19.05 μmol·L^-1 as compared with the normal group. Additionally， the apoptosis rates of the LCA groups （10， 20， 40 μmol·L^-1） significantly increased （<italic>P</italic><0.05）， which reached 30.2% （<italic>P</italic><0.05） at LCA concentration of 40 μmol·L^-1. LCA （10， 20， and 40 μmol·L^-1） decreased the expression of Bcl-2 （<italic>P<</italic>0.05） and increased Bax expression （<italic>P<</italic>0.05） in a dose-dependent manner. Besides， the intracellular ROS level was elevated （<italic>P<</italic>0.05） and mitochondrial MMP was reduced （<italic>P<</italic>0.05） after LCA （10， 20， and 40 μmol·L^-1） treatment in a dose-dependent manner， leading to mitochondrial dysfunction. LCA （10， 20， and 40 μmol·L^-1） induced ER stress to up-regulate the expression of CHOP， ATF4， p-PERK， and p-eIF2<italic>α</italic> （<italic>P<</italic>0.05） in a dose-dependent manner. Conclusion:LCA can induce MDA-MB-231 cell apoptosis by increasing intracellular ROS level and reducing MMP to trigger mitochondrial dysfunction and ER stress.

Shugan Bushen Yulin Decoction Regulates VDAC2 Gene Methylation to Affect Sperm Mitochondrial Function and Improve Sperm Quality in Asthenospermia Rats / 中国实验方剂学杂志

Objective:To investigate the mechanism of Shugan Bushen Yulin decoction in inhibiting voltage-dependent anion-selective channel protein 2 （VDAC2） gene methylation， affecting sperm mitochondrial function， and improving sperm motility through the cyclic adenosine monophosphate/protein kinase A （cAMP/PKA） pathway. Method:Forty male SD rats were randomly divided into the blank group， model group， high- and low-dose Shugan Bushen Yulin decoction groups， and L-carnitine group， with eight rats in each group. Adenine （0.05 g·kg^-1） was administered by gavage for 14 d for inducing oligospermia and asthenospermia. Rats in the Shugan Bushen Yulin decoction groups were treated with intragastric administration of 32.4， 8.1 g·kg^-1Shugan Bushen Yulin decoction， respectively， while those in the L-carnitine group received 0.27 g·kg^-1 L-carnitine by gavage. Following the measurement of sperm motility using an automatic sperm analyzer， the pathological changes in testicular tissue were observed by hematoxylin-eosin （HE） staining. Sperm mitochondrial membrane potential was detected by flow cytometry. The expression of VDAC2 in the testicular tissue was determined by immunofluorescence assay. Real-time polymerase chain reaction （Real-time PCR） was conducted for detecting VDAC2 mRNA expression in testicular tissue. The methylation of VDAC2 gene was examined using bisulfite sequencing. The cAMP expression in testicular tissue was detected by enzyme-linked immunosorbent assay （ELISA）， and the PKA protein expression in testicular tissue by Western blot. Result:Compared with the blank group， the model group exhibited significantly decreased sperm density and motility （<italic>P</italic><0.01）， increased mitochondrial membrane potential （<italic>P</italic><0.01）， down-regulated VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in testicular tissue （<italic>P</italic><0.01）， and elevated VDAC2 gene methylation （<italic>P</italic><0.01）. Compared with the model group， L-carnitine and Shugan Bushen Yulin decoction at the high and low doses all remarkably increased the sperm density and motility and mitochondrial membrane potential （<italic>P</italic><0.01）， up-regulated VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in the testicular tissue （<italic>P</italic><0.01）， and lowered the methylation of VDAC2 in testicular tissue （<italic>P</italic><0.01）. The comparison with the L-carnitine group showed that the sperm density and motility and mitochondrial membrane potential in the low-dose Shugan Bushen Yulin decoction group declined significantly （<italic>P</italic><0.01）. The VDAC2 mRNA and protein expression， PKA protein expression， and cAMP content in the testicular tissue were significantly down-regulated （<italic>P</italic><0.01）， while the methylation of VDAC2 was significantly enhanced （<italic>P</italic><0.01）. Conclusion:Shugan Bushen Yulint decoction may inhibit VDAC2 gene methylation， increase VDAC2 expression， regulate cAMP/PKA pathway， and change mitochondrial membrane potential to enhance the sperm motility.

Construction of mutant NDUFS7 plasmid and its effects on neural cells / 中国药科大学学报

@#To investigate the effects of NDUFS7 gene mutation on neurons, the mutant plasmid of pcDNA3.1(+)-NDUFS7 Q208STOP was constructed and transfected into differentiated SH-SY5Y cells. The effect of transfecting mutant plasmid on the viability of dopaminergic neural cells was detected by MTT assay. The effect of transfection of mutant plasmid on apoptosis was detected by Annexin Ⅴ-FITC/PI staining followed by flow cytometry assay. The changes in the expression levels of apoptosis-related proteins Bax and Bcl-2 in cells after transfection of mutant plasmid were detected by Western blot. The effects of transfection of mutant plasmid on the mitochondrial membrane potential in differentiated SH-SY5Y cells and the intervention effect of antioxidant Trolox were examined using JC-1 fluorescent probe. The intervention effect of Trolox on the apoptosis of differentiated SH-SY5Y cells transfected with mutant plasmid was detected by PI/Hoechst staining. The results showed that the subunit mutation of mitochondrial complex I in dopaminergic neurons could lead to decreased neuronal viability and increased apoptosis, while antioxidants could alleviate the abnormal mitochondrial membrane potential and apoptosis caused by transfection of mutant plasmids, suggesting that transfection of mutant plasmid of NDUFS7 gene could lead to apoptosis by causing abnormal mitochondrial function in dopaminergic neurons.

Inhibitory effect of artesunate on the growth of breast cancer cells and its mechanism / 解放军医学杂志

Objective To investigate the function of artesunate on the growth of breast cancer cell line MDA-MB-231 and its potential mechanism. Methods CCK-8 assays were used to measure growth inhibition in breast cancer cells (MDA-MB-231) in the presence of artesunate, the IC50 was calculated. Based on the IC50 values, various artesunate concentrations (0, 25, 50, 100 μg/ml) were used to experiments in this paper. 0 μg/ml artesunate group served as the control group, using normal saline instead of the artesunate. Apoptosis and mitochondrial membrane potential were analyzed by flow cytometry, after the treatment of various artesunate concentrations (0, 25, 50, 100 μg/ml) for 24 hours. Migration and invasion of the MDA-MB-231 cells were evaluated using wound healing and Transwell assays, respectively, after the treatment of various artesunate concentrations (0, 25 μg/ml) for 24 hours. Results Artesunate inhibited the growth of MDA-MB-231 cells in a dose-dependent manner, with IC50 values for MDA-MB-231 of 54.24 μg/ml. Compared to mock-treated cells, artesunate significantly increased apoptosis, while the mitochondrial membrane potential was significantly decreased after 24 h of exposure to different concentrations of artesunate (P<0.01), in a dose-dependent manner. The cell migration and invasion abilities were also lower in the 25 μg/ml artesunate treated cells than these abilities in the mock-treated cells. Conclusions Artesunate could inhibit the growth of MDA-MB-231 breast cancer cells and inhibit its invasion and migration ability by inducing apoptosis and decreasing mitochondrial membrane potential.

Diethylmorphine effect on action potential and calcium ion current of neonatal rat cardiomyocytes in vitro / 中国组织工程研究

BACKGROUND: Opioids can regulate the changes of membrane potential and Ca2+ current in cardiomyocytes, but whether diacetylmorphine can induce the changes of cardiac rhythm, cell action potential and Ca2+ current has not been reported. OBJECTIVE: To explore the effect of diacetylmorphine on action potential and calcium current of isolated cardiomyocytes from neonatal Sprague-Dawley rats. METHODS: Five concentrations of diacetylmorphine (0, 10-2, 10-3, 10-4, 10-5 mol/L) and 20 mol/L verapamil were used to treat the cardiomyocytes of neonatal Sprague-Dawley rats cultured in vitro. The cells were divided into control group, diacetylmorphine group, diacetylmorphine+verapamil group. The latter two groups were treated with diacetylmorphine and diacetylmorphine+verapamil (20 μmol/L), respectively, while the control group was treated with the same amount of PBS. The study protocol was approved by the Animal Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University on May 21, 2018 with approval No. IACUC201805-K1. RESULTS AND CONCLUSION: At 24 hours of culture with different concentrations of diacetylmorphine, the number of cardiomyocytes with abnormal morphology increased significantly in a dose-dependent manner. When the concentration of diacetylmorphine increased, the number of survived cells decreased, with a reduction in the size of cytoplasm and number of pseudopods, the cell membrane was shrunk and the nuclear structure was blurred. Compared with the control group, when diacetylmorphine was added to intervene with the cardiomyocytes, there was a significant difference in the spontaneous beating frequency and rhythm of cardiomyocytes. The negative value of resting membrane potential decreased, while the time course of action potential increased significantly, and the amplitude of action potential decreased significantly. Compared with the control group, the number of cells with changes in the membrane potential significantly increased in the diacetylmorphine group. The addition of verapamil reduced the number of cells with changes in the membrane potential. Compared with the control group, the number of cells with variation of membrane potential was increased to some extents. These findings suggest that diacetylmorphine can induce cardiomyocyte morphological abnormality, increase the spontaneous beating frequency and rhythm of cardiomyocytes, and change the membrane potential and action potential of cardiomyocytes. Verapamil acts as a calcium channel blocker that can improve the rhythm abnormality of cardiomyocytes induced by diacetylmorphine.

Mitochondrial targeting function of norcantharidin TPP-PEG-PCL nanomicelles promotes apoptosis of liver tumor cells / 中草药

Objective: To prepare norcantharidin TPP-PEG-PCL nanomicelles and study its release in vitro, intracellular transport and promoting effect on hepatoma cell apoptosis. Methods: Thin film hydration method was used to prepare norcantharidin TPP-PEG-PCL nanomicelles, and the particle size, electric potential and microscopic electron microscopy morphological analysis were measured. At the same time, the nanomicelles were evaluated for stability, in vitro release, pharmacokinetics and critical micelle concentration. Coumarin-6 was used as a fluorescent probe to evaluate the uptake of TPP-PEG-PCL nanomicelles in liver tumor cells, lysosomal escape and mitochondrial targeting function; Under the same dosage conditions, the effect of norcantharidin TPP-PEG-PCL nanomicelles on promoting apoptosis of liver tumor cells was evaluated. Results: The cantharidin TPP-PEG-PCL nanomicelles had a particle size of (16.8 ± 0.2) nm, a Zeta potential of (14.3 ± 0.2) mV, and transmission electron microscopy images showed that nanomicelles had a regular spherical shape. The fluorescence test results showed that TPP-PEG-PCL nanomicelles can promote the cellular uptake of drugs, escape lysosomal capture, and finally target aggregation at the mitochondrial site; Cell survival rate and Hoechst staining results showed that cantharidin TPP-PEG-PCL nanomicelles had a good effect on promoting apoptosis of liver tumor cells. Norcantharidin TPP-PEG-PCL nanomicelles can significantly reduce mitochondrial membrane potential, increase intracellular ROS levels, increase pro-apoptotic protein Bcl-2, and reduce resistance. The expression of apoptotic proteins Bax and these pro-apoptotic related experimental results are significantly better than those of norcantharidin PEG-PCL nanomicelles and norcantharidin, which have statistical significance. Conclusion: Norcantharidin TPP-PEG-PCL nanomicelles have good liver tumor cell mitochondrial targeting and promote tumor cell apoptosis, and it is a potentially effective drug delivery system for targeting tumor cell mitochondria.

Repetitive transcranial magnetic stimulation significantly improves cognitive impairment and neuronal excitability during aging in mice / 生物医学工程学杂志

Repetitive transcranial magnetic stimulation (rTMS) is a noninvasive brain stimulation technique that has been paid attention to with increasing interests as a therapeutic neural rehabilitative tool. Studies confirmed that high-frequency rTMS could improve the cognitive performance in behavioral test as well as the excitability of the neuron in animals. This study aimes to investigate the effects of rTMS on the cognition and neuronal excitability of Kunming mice during the natural aging. Twelve young mice, 12 adult mice, and 12 aged mice were used, and each age group were randomly divided into rTMS group and control group. rTMS-treated groups were subjected to high-frequency rTMS treatment for 15 days, and control groups were treated with sham stimulation for 15 days. Then, novel object recognition and step-down tests were performed to examine cognition of learning and memory. Whole-cell patch clamp technique was used to record and analyze resting membrane potential, action potential (AP), and related electrical properties of AP of hippocampal dentate gyrus (DG) granule neurons. Data analysis showed that cognition of mice and neuronal excitability of DG granule neurons were degenerated significantly as the age increased. Cognitive damage and degeneration of some electrical properties were alleviated under the condition of high-frequency rTMS. It may be one of the mechanisms of rTMS to alleviate cognitive damage and improve cognitive ability by changing the electrophysiological properties of DG granule neurons and increasing neuronal excitability.

MiR-484 Protects Rat Myocardial Cells from Ischemia-Reperfusion Injury by Inhibiting Caspase-3 and Caspase-9 during Apoptosis

BACKGROUND AND OBJECTIVES: To reveal the detail mechanism of miR-484 on myocardial ischemia-reperfusion (MI/R) injury.METHODS: Rats model of MI/R injury was established based on control (Con; sham operate) group, ischemia-reperfusion (I/R) group, miR-484 treatment (miR) group, and I/R-negative control (IR-C) group, followed by pathological and interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β expression evaluation. Then the myocardial apoptosis, as well as the expression of miR-484, caspase-3, and caspase-9 in myocardium were examined. Finally, the regulatory relation between miR-484 and SMAD family member 7 (SMAD7) was predicated, followed by verification analysis.RESULTS: Compared with Con group, the expression of miR-484 in I/R and IR-C group was decreased. Compared with I/R and IR-C group, the expression of miR-484 was increased in miR group. Compared with Con group, the expression levels of IL-6, TNF-α, and IL-1β in cardiac myocytes of I/R group and IR-C group were increased. Compared with Con group, the apoptotic index, membrane potential of I/R, and the expression of caspase-3/9 were increased in IR-C group. Compared with the I/R and IR-C groups, the apoptotic index of myocardial cells in the ischemic region was decreased, the membrane potential was increased, and the expression of caspase-3/9 was decreased significantly in the miR group. SMAD7 was the target gene of miR-484.CONCLUSIONS: MiR-484 protected myocardial cells from I/R injury by suppressing caspase-3 and caspase-9 expression during cardiomyocyte apoptosis. MiR-484 reduced the expression of IL-6, TNF-α, and IL-1β in MI/R. MiR-484 might alleviate the decreasing of mitochondrial membrane potential in MI/R cells.

Serum Electrolytes and Red Blood Cell Membrane Potential of Hypertensive Patients

Serum electrolyte and red blood cell membrane potential of hypertensive patients in Owerri metropolis were investigated. A total of 50 volunteer subjects were used for the study. Thirty (30) of the volunteer subjects were hypertensive subjects and were used as test subjects; while the remaining 20 subjects were healthy subjects with normal blood pressure used as normentensive subjects (control). Results observed showed increased red blood cell (RBC) K+ and Cl- in hypertension subjects against normentensive subjects. Apart from K+ which reduced significantly (p<0.05), other electrolyte ions of the serum increased significantly (p<0.05) in hypertensive subjects against normentensive subjects. However, Na+ and Cl- membrane potential was not significantly (p>0.05) altered in hypertensive subjects against normotensive subjects while K+ was significantly (p<0.05) altered. The observed alterations in the parameters investigated in hypertensive subjects in the present study could be as a result of a host of derangements involving electrolyte metabolism, altered membrane transport and a possible increase in membrane fragility. This study has shown the serum electrolyte and red blood cell membrane potential of hypertensive patients in Owerri metropolis.

Effects of glutamine deprivation on oxidative stress and cell survival in breast cell lines

BACKGROUND: Tumourigenic cells modify metabolic pathways In order to facilitate increased proliferation and cell survival resulting in glucose-and glutamine addiction. Previous research indicated that glutamine deprivation resulted in potential differential activity targeting tumourigenic cells more prominently. This is ascribed to tumourigenic cells utilising increased glutamine quantities for enhanced glycolysis-and glutaminolysis. In this study, the effects exerted by glutamine deprivation on reactive oxygen species (ROS) production, mitochondrial membrane potential, cell proliferation and cell death in breast tumourigenic cell lines (MCF-7, MDA-MB-231, BT-20) and a non-tumourigenic breast cell line (MCF-10A) were investigated. RESULTS: Spectrophotometry demonstrated that glutamine deprivation resulted in decreased cell growth in a time-dependent manner. MCF-7 cell growth was decreased to 61% after 96 h of glutamine deprivation; MDA-MB-231 cell growth was decreased to 78% cell growth after 96 h of glutamine deprivation, MCF-10A cell growth was decreased 89% after 96 h of glutamine deprivation and BT-20 cell growth decreased to 86% after 24 h of glutamine deprivation and remained unchanged until 96 h of glutamine deprivation. Glutamine deprivation resulted in oxidative stress where superoxide levels were significantly elevated after 96 h in the MCF-7-and MDA-MB-231 cell lines. Time-dependent production of hydrogen peroxide was accompanied by aberrant mitochondrial membrane potential. The effects of ROS and mitochondrial membrane potential were more prominently observed in the MCF-7 cell line when compared to the MDA-MB-231-, MCF-10A- and BT-20 cell lines. Cell cycle progression revealed that glutamine deprivation resulted in a significant increase in the S-phase after 72 h of glutamine deprivation in the MCF-7 cell line. Apoptosis induction resulted in a decrease in viable cells in all cell lines following glutamine deprivation. In the MCF-7 cells, 87.61% of viable cells were present after 24 h of glutamine deprivation. CONCLUSION: This study demonstrates that glutamine deprivation resulted in decreased cell proliferation, time-dependent- and cell line-dependent ROS generation, aberrant mitochondrial membrane potential and disrupted cell cycle progression. In addition, the estrogen receptor positive MCF-7 cell line was more prominently affected. This study contributes to knowledge regarding the sensitivity of breast cancer cells and non-tumorigenic cells to glutamine deprivation.

Inhibitory effect of isolinderalactone on growth of human breast cancer MCF-7 cells and its mechanism / 中草药

Objective: To study the effects and the mechanism of isolinderalactone on inhibiting the growth of human breast cancer MCF-7 cells. Methods: Human breast cancer MCF-7 cells were cultured in vitro and treated respectively with indicated concentrations of isolinderalactone by cell culture technique. The proliferation rate was detected by MTT method; Flow cytometry and TUNEL assay were used to observe the effects of isolinderalactone on cell cycle, mitochondrial membrane potential and apoptosis in MCF-7 cells; The apoptosis related protein expression levels were determined by Western blotting. Results: Isolinderalactone significantly inhibited the proliferation of MCF-7 cells by inducing cell apoptosis in a time and concentration dependent manner and induced cell cycle arrest at G2/M phases. The mitochondrial membrane potential was depolarized; And isolinderalactone up-regulated the expressions of apoptosis related proteins Bax and cleaved Caspase-3 and down-regulated the expressions of apoptosis related proteins Bcl-2. Conclusion: Isolinderalactone shows obvious anti-cancer activities by inducing cell apoptosis. The mechanism of inducing apoptosis may be associated with the reduction of mitochondrial membrane potential and activation of caspase pathway.