Role of mitochondrial fusion and fission in protective effects of dexmedetomidine against cerebral ischemia/reperfusion injury in mice / 南方医科大学学报

OBJECTIVE@#To investigate the protective effects of dexmedetomidine (DEX) against cerebral ischemia/reperfusion (I/R) injury in mice and its relation with mitochondrial fusion and fission.@*METHODS@#Male ICR mice were randomly divided into sham-operated group, I/R group, I/R+DEX group and I/R+DEX+dorsomorphin group. Mouse models of cerebral I/R injury were established by modified thread occlusion of the middle cerebral artery. DEX (50 μg/kg) was injected intraperitoneally at 30 min before cerebral ischemia, which lasted for 1 h followed by reperfusion for 24 h. The neurobehavioral deficits of the mice were evaluated based on Longa's scores. The volume of cerebral infarction was detected by TTC staining. The changes in mitochondrial morphology of the brain cells were observed with transmission electron microscopy. Western blotting was performed to detect the expressions of phosphorylated AMP-activated protein kinase (p-AMPK), mitochondrial fusion protein (Mfn2) and mitochondrial fission protein (p-Drp1) in the brain tissues.@*RESULTS@#DEX pretreatment significantly reduced the neurobehavioral score and the percent volume of cerebral infarction in mice with cerebral I/R injury. Treatment with dorsomorphin (an AMPK inhibitor) in addition to DEX significantly increased the neurobehavioral score and the percent volume of cerebral infarction in the mouse models. Transmission electron microscopy showed that DEX obviously reduced mitochondrial damage caused by cerebral I/R injury and restored mitochondrial morphology of the brain cells, and such effects were abolished by dorsomorphin treatment. Western blotting showed that DEX pretreatment significantly increased the expressions of p-AMPK and Mfn2 protein and decreased the expression of p-Drp1 protein in the brain tissue of the mice, and these changes were also reversed by dorsomorphin treatment.@*CONCLUSIONS@#Preconditioning with DEX produces protective effects against cerebral I/R injury in mice possibly by activating AMPK signaling to regulate mitochondrial fusion and fission in the brain cells.

Down-regulation of pannexin 2 channel enhances cisplatin-induced apoptosis in testicular cancer I-10 cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of down-regulation of pannexin 2 (Panx-2) channels on cisplatin-induced apoptosis in I-10 cells.@*METHODS@#The expression of Panx-2 protein in testicular cancer cells was detected with Western blotting. The testicular cancer cell line I-10 was transfected with two short hairpin RNA (shRNA1 and shRNA2) Lipofectamine, the empty vector (NC group) or Lipofectamine2000 (blank control group), and the changes in the expression of Panx-2 was detected with Western blotting. The effects of transfection with a Panx-2 inhibitor on surviving fraction of the cells treated with cisplatin (16 μmol/L) for 24 h, 48 h and 72 h was assessed with MTT assay, and the clonogenic capacity of the cells was evaluated with colony-forming assay. At 8 h after incubation with 16 μmol/L cisplatin, AnnexinV/PI double staining was used to detect the early apoptosis of the cells. After 24 h of treatment with 16 μmol/L cisplatin, the cells were examined for expressions of caspase-3, Bcl-2 and Bax using Western blotting.@*RESULTS@#The expression of Panx-2 was significantly increased in cisplatin-resistant I-10/DDP ( < 0.001) cells and Tcam-2/DDP ( < 0.01) cells as compared with I-10 cells and Tcam-2 cells. Transfection of I-10 cells with shRNA1 and shRNA2 resulted in significantly decreased Panx-2 expression ( < 0.05) and significantly reduced cell surviving fraction ( < 0.001). In the presence of cisplatin, the cells in NC group showed a higher clonogenic efficiency than those in shRNA1 and shRNA2 groups ( < 0.001). The early-stage apoptosis rate of the cells in shRNA1 and shRNA2 groups were significantly higher than that in NC group ( < 0.01). Panx-2 knockdown in I-10 cells significantly increased caspase-3 and Bax expressions ( < 0.05) and significantly decreased the expression of Bcl-2 ( < 0.01).@*CONCLUSIONS@#Down-regulation of Panx-2 channel enhances cisplatin-induced apoptosis in cultured testicular cancer cells.

SRC kinase inhibitor PP2 inhibits invasion and metastasis of lung cancer A549 cells by upregulating connexin43 expression / 南方医科大学学报

OBJECTIVE@#To investigate the effect of SRC kinase inhibitor PP2 on the invasion and metastasis of lung cancer A549 cells and explore its molecular mechanism.@*METHODS@#MTT assay was used to evaluate the inhibitory effect of PP2 on the proliferation of A549 cells. Cell scratch and Transwell assays were performed to assess the invasion and metastatic capacity of A549 cells after treatment with 1, 2, 4, 8, and 16 μmol/L PP2 for 24 h. Western blotting was used to detect the expressions of connexin43 (Cx43) and MMP-2 in the cells after small interfering RNA (siRNA)-mediated silencing or overexpression of Cx43; the changes in the cell invasion and metastasis in response to PP2 treatment after Cx43 silencing or overexpression were investigated.@*RESULTS@#MTT assay showed that treatment with PP2 at 2, 4, 8, 16, and 32 μmol/L significantly inhibited the proliferation of A549 cells in a concentration-dependent manner. Treatments with PP2 at 1, 2, 4, 8, and 16 μmol/L for 24 h also concentration-dependently lowered the invasion and metastatic abilities of the cells ( < 0.05). At 4 and 8 μmol/L, PP2 significantly increased the expression level of Cx43 protein and decreased the expression level of MMP-2 protein. Overexpression of Cx43 significantly enhanced the inhibitory effect of PP2 on the cell invasion and metastasis, and Cx43 silencing significantly attenuated the inhibitory effect of PP2 ( < 0.05).@*CONCLUSIONS@#PP2 treatment can suppress the invasion and metastasis of A549 cells possibly by modulating the expression of Cx43.

Inhibitory effect of connexin43 protein on autophagy in cisplatin-resistant testicular cancer I-10 cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of connexin43 (Cx43) protein on autophagy in cisplatin (DDP)-resistant testicular cancer I-10 cells.@*METHODS@#The expression of Cx43 proteins in testicular cancer I-10 cells and I-10/DDP cells were detected with Western blotting. I-10/DDP cells were transfected with a full- length mouse Cx43 vector (mCx43) Lipofectamine, the empty vector or Lipofectamine (blank control group), and the changes in the expressions of LC3 and p62 proteins were determined with Western blotting. mCherry-GFP-LC3B transfection and transmission electron microscopy were used to analyze the changes in autophagy of the cells with Cx43 overexpression.@*RESULTS@#Cx43 was significantly decreased in I-10/DDP cells compared with I-10 cells ( < 0.01). Transfection of the I-10/DDP cells with mCx43 vector resulted in significantly increased Cx43 expression in the cells ( < 0.01) and caused significantly decreased expression of LC3-Ⅱ ( < 0.01) and increased expression of p62 ( < 0.05) as compared with the negative control cells. Both transmission electron microscopy and mCherry-GFP-LC3B transfection showed that the number of autophagosomes was obviously reduced in mCx43-transfected cells as compared with the negative control cells.@*CONCLUSIONS@#Cx43 inhibits autophagy in cisplatin-resistant testicular cancer I-10 /DDP cells.

Protective effects of P2X7 receptor inhibition in cerebral ischemia/reperfusion injury in rats / 中南大学学报(医学版)

To investigate the protective effects of P2X7 receptor (P2X7R) inhibitor against cerebral ischemia/reperfusion (I/R) injury in rats and the possible mechanisms.
 Methods: The neurological deficit of rats was evaluated by Longa score. The infarct volume was examined by 2, 3, 5-triphenyltetrazolium chloride (TTC) staining. The expression levels of extracellular signal-regulated kinase (ERK), phosphorylation extracellular signal-regulated kinas p-ERK), connexin 43 (Cx43), Bax, Bcl-2 and cleaved caspase-3 were detected by Western blot.
 Results: Compared with sham group, the neurobehavioral score (P<0.05) and cerebral infarct volume (P<0.01) of rats in I/R group was increased. Compared with I/R group, brilliant blue G (BBG, the antagonist of P2X7R) or PD98059 (the inhibitor of EKR kinase) could reduce the neurobehavioral score (P<0.01) and cerebral infarct volume significantly (P<0.05). The neurobehavioral score and cerebral infarct volume was further decreased (P<0.05) when rats were treated with both BBG and PD98059. Compared with I/R group, the expression levels of p-ERK, Cx43, cleaved caspase-3 and the ratio of Bax/Bcl-2 were decreased by BBG or PD98059 pretreatment, and the protective effects were further enhanced when rats were treated with both BBG and PD98059 (P<0.05).
 Conclusion: Inhibition of P2X7R reduces the cerebral I/R injury of rats. ERK inhibition is probably involved in the protective effects of P2X7R inhibitor against cerebral I/R injury, which may be related to the reduction of Cx43 and cleaved caspase-3, and the ratio of Bax/Bcl-2.

Protective effect of NADPH oxidase against cerebral ischemia/reperfusion injury / 中国药理学通报

Aim To determine the protective effect of NADPH oxidase against focal cerebral ischemia/reper-fusion ( I/R) injury in rats. Method A thread occlu-sion method was used to make a middle cerebral artery occlusion ( MCAO ) model. Apocynin ( 2. 5 mg · kg-1 ) was injected by tail vein 15 min before ischemi-a. Then, the neurological behavior, cerebral infarction volume, pathological morphological changes and the expression of Cx36, PKC, Bax, Bcl-2 of rats were de-tected after ischemia for 2 h, followed by reperfusion for 24 h. Results Compared with cerebral I/R group, administration of apocynin significantly reduced the neurological behavior scores and the cerebral in-farction volume percentage, alleviated the pathological morphological damage, increased the protein expres-sion of Cx36 and PKC, and reduced the Bax/Bcl-2 ra-tio of rats with focal cerebral I/R injury. Compared with apocynin group , apocynin combined with PKC inhibitor significantly reduced above protective effects. Conclusion Inhibition of NADPH oxidase could alle-viate cerebral I/R injury, increase the levels of Cx36, PKC proteins and reduce the Bax/Bcl-2 ratio.

Src kinase inhibitor PP2 protects rat astrocytes from hypoxia/reoxygenation injury in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of Src kinase inhibitor PP2 on hypoxia/reoxygenation (H/R) injury in rat astrocytes in vitro.</p><p><b>METHODS</b>In vitro cultured rat astrocytes were exposed to hypoxia for 8 h followed by reoxygenation for 24 h with or without pretreatment with PP2 (10 µmol/L) for 24 h before H/R injury. MTT assay and flow cytometry were used to detect the viability and apoptosis of the exposed astrocytes, respectively, and the protein expressions of Src, Bax, and Bcl-2 in the cells were determined using Western blotting.</p><p><b>RESULTS</b>PP2 pretreatment significantly increased the viability and decreased the apoptosis rate of rat astrocytes exposed to H/R injury (P<0.01). Western blotting showed that H/R injury caused increased expression of Src kinase, which was lowered by PP2 pretreatment. The ratio of Bax/bcl-2 in the astrocytes increased after H/R injury, and was significantly decreased by PP2 pretreatment (P<0.01).</p><p><b>CONCLUSION</b>PP2 protects rat astrocytes from H/R injury possibly by inhibiting the expression of Src kinase and activating the anti-apoptotic mechanisms in the cells.</p>

Protective effect of propofol against cerebral ischemic/reperfusion injury may involve inhibition of gap junction / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the protective effect of propofol against focal cerebral ischemia/reperfusion (I/R) injury in rats and its relation with gap junction.</p><p><b>METHODS</b>Seventy adult male SD rats were randomly divided into sham-operated group, I/R group, low-, moderate-, and high-dose propofol groups (25, 50, 100 mg/kg; P25, P50, P100 groups, respectively), I/R+CBX group and P100+CBX group. Thread occlusion was used to induce middle cerebral artery occlusion (MCAO) in the mice for 2 h followed by reperfusion for 24 h. Longa's scores were used to evaluate the neurological behavior of the rats. TTC staining was used to measure the cerebral infarction volume and Western blotting was performed to detect the expressions of Cx43, PKC, Bax, and Bcl-2 in the brain of the rats.</p><p><b>RESULTS</b>Compared with the I/R group, the rats pretreated with moderate and high doses of propofol showed significantly reduced neurological behavior scores and cerebral infarction volume percentage, and the effect was more obvious in high-dose propofol pretreatment group. CBX obviously enhanced the protective effect of propofol against I/R injury. Compared with those in the sham-operated group, the protein expression of Cx43 and the Bax/Bcl-2 ratio were increased and the protein expression of PKC was reduced in I/R group, and these changes were significantly reversed by high-dose propofol pretreatment; the effects of propofol were further enhanced by CBX.</p><p><b>CONCLUSION</b>The protective effect of propofol against cerebral I/R injury may involve the inhibition of the gap junction via PKC signaling and by reducing the Bax/Bcl-2 ratio.</p>

Comparison of cisplatin-resistant testicular cancer cell lines established by two methods / 南方医科大学学报

<p><b>OBJECTIVE</b>To compare the biological behaviors of two drug-resistant testicular cancer cell lines established by different methods.</p><p><b>METHODS</b>Drug-resistance was induced in testicular cancer cell lines exposure of the cells to increasing concentrations of or a high dose of cisplatin (I-10/DDPi and I-10/DDPh cell lines, respectively). The morphological characteristics of the two cell lines were observed microscopically. The resistance index of the cells was determined with MTT assay, and the cell growth curves were drawn. The cellular expression of resistance-associated proteins MDR1 and P-gp was detected by Western blotting. The cell invasion ability was assessed with Transwell assay.</p><p><b>RESULTS</b>Normal testicular cancer cell line I-10 and the two resistant cell lines all showed an adherent growth pattern. Compared with I-10 cells, I-10/DDP cells exhibited slightly heterogenous cell sizes, irregular shapes, the presence of microvilli tentacles on the cell surface, and a scattered arrangement. The cisplatin resistance index of I-10/DDPi and I-10/DDPh cells were 3.924 and 3.099, respectively. Compared with I-10, the drug-resistant cell lines showed extended doubling time with increased expressions of MDR1 and P-gp and increased cell invasiveness, which was especially obvious in I-10/DDPi cells.</p><p><b>CONCLUSION</b>Both increasing dose exposure and high-dose exposure to cisplatin can induce cisplatin resistance in testicular cancer cells, and the resistant cells established by the latter method better mimics clinical drug-resistant tumor cells.</p>

Functional enhancement of gap junction by valproate acid sodium in breast cancer cells and the mechanism / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of valproate acid sodium (VPA) on gap junction intercellular communication in breast cancer Hs578T cells and explore the mechanism.</p><p><b>METHODS</b>MTT assay was used to detect the cytotoxicity of VPA on Hs578T cells, and parachute assay was used to detect the effect of VPA on dye spread of the cells. Western blotting was employed to detect the expression changes of Cx43 total protein in VPA-treated Hs578T cells. The effect of VPA on the expression of Cx43 on the surface of Hs578T cells was examined with immunofluorescence assay.</p><p><b>RESULTS</b>MTT assay showed no obvious cytotoxicity of VPA on Hs578T cells at the concentrations below 10 mmol/L. VPA below 5 mmol/L obviously increased the gap junction function in Hs578T cells (P<0.01), and significantly enhanced the expression of Cx43 total protein (P<0.01) and Cx43 expression on the surface of Hs578T cells (P<0.01).</p><p><b>CONCLUSION</b>VPA can obviously increase the gap junction function in Hs578T cells possibly by enhancing Cx43 total protein expression and Cx43 protein expression on the surface of Hs578T cells.</p>

Expression of connexin 43 and functional modulation of gap junction in neonatal rat astrocytes in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To determine the expression of connexin 43 (Cx43) protein and explore the functional modulation of gap junction intercellular communication in astrocytes.</p><p><b>METHODS</b>Cultured neonatal SD rat astrocytes were divided into normal control group, all-trans retinoic acid (ATRA) group (treated with 10 µmol/L ATRA for 24 h) and oleamide group (treated with 25 µmol/L oleamide for 2 h). Western blotting and immunofluorescence assay were used to detect total cellular Cx43 protein expression and Cx43 expression on the surface of the astrocytes, respectively. Parachute assay was used to evaluate the functional changes of gap junction intercellular communication of the astrocytes.</p><p><b>RESULTS</b>Compared with the normal control cells, ATRA treatment resulted in a significantly increased expression of total Cx43 protein in the astrocytes (P<0.01), and oleamide significantly suppressed its expression (P<0.01). Similarly, ATRA obviously enhanced while oleamide suppressed Cx43 protein expression on the surface of the astrocytes. The gap junction intercellular communication of the astrocytes was enhanced by ATRA (P<0.01) and inhibited by oleamide (P<0.01).</p><p><b>CONCLUSION</b>ATRA and oleamide can modulate gap junction intercellular communication of the astrocytes possibly by regulating the expression of Cx43 protein.</p>

Protective effects of pomegranate polyphenols on cardiac function in rats with myocardial ischemia/reperfusion injury / 南方医科大学学报

<p><b>OBJECTIVE</b>To determine the protective effect of pomegranate polyphenols on cardiac function in rats with myocardial ischemia/reperfusion (I/R) injury and explore the possible mechanism.</p><p><b>METHODS</b>Fifty SD rats were randomized into 5 equal groups, including a sham-operated group, an I/R model group, and 3 pomegranate polyphenol dose groups (150, 300, and 600 mg/kg). The rats were subject to a 45-min left main coronary artery occlusion followed by a 180-min reperfusion to induce myocardial I/R injury except for the rats those in the sham-operated group. The cardiac functions were monitored continuously during the experiment. At the end of the reperfusion, arterial blood samples were obtained to measure plasma contents of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA) and the activities of superoxide dismutase (SOD). Triphenyltetrazolium chloride (TTC) staining was used to evaluate the infarct size.</p><p><b>RESULTS</b>Compared with the sham-operated group, all the rats with I/R showed significantly decreased left ventricular systolic pressure (LVSP), maximal rates of increase/decrease of the left ventricular pressure (±dp/dtmax) (P<0.01) and significantly increased left ventricular end-diastolic pressure (LVEDP) (P<0.01). Compared with the I/R model group, all the 3 pomegranate polyphenol groups had significantly improved cardiac function (P<0.05), decreased plasma contents of CK, LDH and MDA (P<0.01), increased SOD activities (P<0.01), and obviously reduced infarct size (P<0.01).</p><p><b>CONCLUSION</b>Pomegranate polyphenols can protect the cardiac function of rats with I/R injury probably in association with their actions in enhancing oxygen free radical scavenging activity and decreasing lipid peroxidative damage of the myocardial tissues.</p>

Effects of Bingdouting Capsule on Heart Rate and Sinoatrial Node Function in Rabbit Model of Sick Sinus Syndrome / 中药新药与临床药理

Objective To observe the effect of Bingdouting capsule(BC)on rabbit model of sick sinus syndrome.Methods Rabbit models of sick sinus syndrome were established by external application of 20 % methanal on the region of sinoatrial node.The effect of BC on heart rate and electrophysicological parameters of sinoatrial node was observed.Results BC markedly increased the heart rate,shortened the sinoatrial conduction time(SACT)and corrected sinoatrial node recovery time(CSNRT).Conclusion BC has an obvious effect for the treatment of sick sinus syndrome in rabbits.

Inhibitory effects of heparan sulfate proteoglycan on transplanted breast cancer in C_3H mice / 中国药理学通报

Aim To observe the anti-tumor activity and the mechanism of heparan sulfate proteoglycan(HSPG) on C3H mice transplanted tumors.Methods Tumor model was established and randomly divided into five groups.HSPG groups(5,10,50 mg?kg-1),positive group and control group,intraperitoneal injection was performed once a day for 22 days and the volume of tumors was measured.Mice were treated on 24th day,then tumor weight was examined,thymus index,and spleen index were calculated,the apoptosis was determined by TdT-mediated Dutp nick end labeling(TUNEL) assay in situ,the expression of vascular endothelial growth factor(VEGF) was detected by immunohistochemistry.Results The tumor volume in HSPG groups was reduced without the decrease of thymus index,spleen index.TUNEL assay in situ showed numerous heavy blue apoptosis cells in the HSPG groups significantly higher than in control groups.The tumors in HSPG groups showed significantly lower VEGF expression than those in control group.Conclusion HSPG had significant anti-tumor effects on C3H mice transplantable breast cancer.The mechanisms may be associated with the effects of inducing tumor cell apoptosis and inhibiting the VEGF expression.