Preliminary mechanism of senegenin against H/R-induced apoptosis of primary cortical neurons

AIM：To explore the preliminary mechanism of senegenin (Sen) on inhibiting hypoxia/reoxygenation(H/R)-induced apoptosis of primary cortical neurons. METHODS：The cultured cortical neurons were randomly divided into normal group (control group), model group (H/R group), Sen+H/R group and Sen group. Flow cytometry was used to evaluate the effect of Sen on H/R-induced cell apoptosis. The protein levels of JNK, p-JNK, c-Jun, p-c-Jun, Bcl-2 and Bax were assessed by Western blotting. RESULTS：The apoptotic rate in H/R group was obviously higher than that in control group (P<0.05), while the apoptotic rate in Sen+H/R group was obviously lower than that in H/R group (P<0.05), suggesting that the model of apoptosis was established successfully. The results of Western blotting showed that Sen increased the expression of JNK and c-Jun, inhibited the phosphorylation of JNK and c-Jun (P<0.05), increased the protein level of Bcl-2 and inhibited the protein level of Bax in H/R treated primary cortical neurons (P<0.05). CONCLUSION：Sen has a protective effect against H/R-induced neuronal apoptosis by increasing the expression of JNK and c-Jun, inhibiting the phosphorylation of JNK and c-Jun, increasing the protein level of Bcl-2 and decreasing the protein level of Bax.     

PSMA activates JNK/SAPK pathway and regulates apoptosis of prostate cancer cells

AIM：To investigate the effects of prostate-specific membrane antigen (PSMA) on the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) pathway and the apoptosis of prostate cancer cells. METHODS：The shRNA lentiviral vector with high efficiency was constructed in the previous study to block the PSMA expression in the prostate cancer cells as experimental interference group, while the constructed vector of PSMA was transfected into the prostate cancer cells to promote PSMA expression as positive experimental group. The control group was the cell line without any treatment. JNK/SAPK inhibitor SP600125 was used as a negative control. Western blotting and immunocytochemistry were used to observe the p-JNK/SAPK expression. The cell growth curve was determined by CCK-8 assay. The cell cycle and apoptosis were analyzed by flow cytometry. RESULTS：Inhibition of PSMA expression resulted in the decrease of p-JNK/SAPK expression levels, while enhancement of the PSMA expression made the increase in the expression of p-JNK/SAPK. SP600125 decreased the level of p-JNK/SAPK, and no significant difference among the 3 groups was observed. The cell proliferation and S-phase percentage decreased after the inhibition of PSMA, while the cells in the 3 groups with SP600125 treatment only had low levels of cell proliferation and percentage of S phase. The inhibition of PSMA promoted apoptosis, while in the enhanced PSMA expression group, apoptotic rate was significantly reduced. After adding SP600125, the cell apoptotic rate was lower than that in normal culture group. CONCLUSION：PSMA has an impact on the proliferation, cell cycle and apoptosis of prostate cancer cells by up-regulating JNK/SAPK signaling pathway, but the JNK/SAPK signaling is not the only path.     

Oxidative stress-activated JNK-MAPK signaling pathway is involved in fluctuant high glucose-induced injury of hepatocytes

AIM: To explore the mechanisms of fluctuant high blood glucose-induced apoptosis of hepatocytes. METHODS: SD rats were randomly divided into normal control group (N), stable high blood glucose group (S), fluctuant high blood glucose group (F) and insulin group (I). Diabetic rats were induced by intraperitoneal injection of streptozotocin (65 mg/kg), and the fluctuant high blood glucose animal model was induced by intraperitoneal injection of ordinary insulin and glucose at different time points every day. The blood glucose fluctuation patterns of the animals in F group within 12 weeks were similar every day and no significant difference of the HbA1c concentration was observed compared with S group, indicating that the fluctuant hyperglycemia was successfully established in F group. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the content of malondialdehyde (MDA) and nitric oxide (NO) in the homogenate of the liver tissues were detected by colorimetry. The mRNA and protein levels of JNK, p-JNK, Bax and Bcl-2 were examined by RT-PCR and Western blot. RESULTS: After 12 weeks, the increases in the intakes of food and water, the urine output, and the abnormal liver function were observed in S group, I group and F group. Compared with N group, the MDA level was increased, the content of NO and the activity of SOD and GSH-Px were decreased, and up-regulation of JNK mRNA and p-JNK and Bax proteins, and down-regulation of Bcl-2 were also found in S group, I group and F group. The above effects were more obviously showed in F group. CONCLUSION: Oxidative stress activates JNK-MAPK signaling pathway, which is involved in fluctuant high glucose-induced apoptosis of hepatocytes.     

Relationship between histone H3K36 trimethylation and acute renal injury induced by ischemia/reperfusion

AIM: To observe the expression change of H3K36 trimethylation (H3K36me3) in acute kidney injury (AKI) induced by ischemia/reperfusion (IR) and analyze its correlation with SMYD2 and renal injury, and to provide a new target for clinical treatment of IR-AKI. METHODS: The ICR mice (n=30) were randomly divided into IR group (n=15) and sham operation group (sham group, n=15). The AKI model was established by clamping bilateral renal pedicles for 45 min. The serum and renal tissues of the mice were collected after the model was established for 24 h. The levels of blood urea nitrogen (BUN) and serum creatinine (SCr) were detected by biochemical method. The pathological changes of the kidney were observed by HE staining. The changes of NGAL and cleaved caspase-3 were observed by immunohistochemical (IHC) staining. The protein levels of NGAL, SMYD2, H3K36me3, p-P53, P53, cleaved caspase-3, Bax, Bcl-2, STAT3, p-STAT3, JNK and p-JNK1/2/3 in the renal tissues were determined by Western blot. The relationship between H3K36me3 and SMYD2/NGAL was analyzed by Pearson correlation method. RESULTS: Compared with sham group, BUN and SCr levels of the mice in IR group were significantly increased (P<0.05). HE staining showed significant edema, abscission and necrosis in renal tubular epithelial cells of the micc in IR group, and abscission of brush border and large amount of cell debris in the lumen were also observed. The IHC staining results showed that the protein levels of NGAL and cleaved caspase-3 in IR group were significantly increased. The results of Western blot showed that the protein levels of NGAL, SMYD2, H3K36me3, p-P53, cleaved caspase-3, Bax, STAT3, p-STAT3, JNK and p-JNK1/2/3 in IR group were significantly up-regulated (P<0.05), while the Bcl-2 levels were significantly down-regulated (P<0.05). The correlation analysis showed that H3K36me3 was positively correlated with the levels of SMYD2 and NGAL. CONCLUSION: H3K36me3 is closely related to SMYD2 and renal injury in IR-AKI mice, and the up-regulated expression of H3K36me3 may be involved in the regulation of IR-AKI occurrence and development together with the activation of STAT3 and JNK signaling pathways.     

Effect of low molecular weight heparin on hypoxia-induced apoptosis in primary cultured neonate rat cerebral cortical neurons

AIM:To investigate the mechanism by which low molecular weight heparin (LMWH) inhibits apoptosis of primary cultured cerebral cortical neurons caused by hypoxia. METHODS:The anti-apoptosis effect of LMWH on primary cultured neurons was observed by methods of the primary culture of cerebral neurons of postnatal rats in free-serum with neurobasal medium supplied with 2% B27 supplement, hypoxic culture of neurons, Hoechst 33342 staining and immunocytochemistry. RESULTS:At concentrations of 250-500 U/L, LMWH reduced apoptosis rate of cerebral cortical neurons induced by hypoxia (P<0.05) and apoptosis rate was lower in LMWH groups than that in BDNF (50 μg/L) group (P<0.05). LMWH (500 U/L) increased Bcl-2 protein expression (P<0.05) and decreased Bax protein expression (P<0.01) in the cerebral cortical neurons induced by hypoxia, the ratio of Bcl-2/Bax was improved (P<0.01). The ratio of Bcl-2/Bax in LMWH (500 U/L) group was higher than that in normal control group, BDNF group and apoptosis positive group (P<0.05). CONCLUSION:LMWH at concentrations of 250-500 U/L is able to prevent cerebral cortical neurons from apoptosis in primary culture under hypoxia. The effect of anti-apoptosis may be related to up-regulation of Bcl-2 protein expression, down-regulation of Bax-2 protein expression, and increase in the ratio of Bcl-2/Bax.     

Lycopene protects primary mouse cerebrocortical neurons against t-BHP-induced damage in vitro

AIM: To investigate the protective effect of lycopene on primary mouse cerebrocortical neurons exposed to tert-butyl hydroperoxide (t-BHP) and its mechanisms of in vitro.METHODS: Primary cerebrocortical neurons of newborn C57 mice were extracted and divided into normal group, t-BHP group, lycopene+t-BHP group and lycopene group. The neuronal damage was induced by t-BHP exposure for 24 h, and the cell viability was examined by MTT assay. ROS content was measured by flow cytometry, and the protein levels of Bax, Bcl-2, caspase-3, cleaved caspase-3 and cytochrome C were examined by Western blot.RESULTS: The primary mouse cortical neurons expressed MAP-2 protein. Lycopene at concentration of 4 μmol/L reversed the decrease in cell viability. Flow cytometry revealed that lycopene treatment attenuated ROS content under the condition of t-BHP exposure. In addition, the protein level of Bcl-2 was increased, and the expression of Bax, cleaved caspase-3 and cytochrome-C was suppressed in lycopene+t-BHP group.CONCLUSION: The protective effect of lycopene on cortical neurons with t-BHP-induced injury may be involved in the mechanism of neuronal antioxidative response by down-regulating caspase-3 and Bax/Bcl-2 through the mitochondrial apoptotic pathway.     

Cardiomyocyte apoptosis and related gene expression after acute myocardial infarction in rats

AIM: To investigate cardiomyocyte apoptosis and the expression of caspase-3, Bcl-2 and Bax after acute myocardial infarction (AMI) in rats.METHODS: AMI model was established with the ligation of left coronary artery in 78 randomly selected female SD rats.Twenty-four hours after operation, 43 survivors were randomly divided into 48-hour and 4-week two groups according to the time points: MI 48 h (n=11) and MI4 weeks (n=13) groups, sham-operated rats (S, n=27) were also randomly selected and reassigned to S48 h (n=10) and S4 weeks (n=10) groups.Cardiomyocyte apoptosis was detected with in situ terminal deoxynucleotidyl transferase (TdT)-dUTP nick-end labeling (TUNEL staining) and DNA gel electrophoresis.Caspase-3, Bcl-2 expression and Bax expression were detected with immunohistochemistry and Western blotting analysis.RESULTS: Compared with sham-operated group, after AMI, systolic, diastolic, and mean arterial blood pressures (SBP, DBP, MAP), left ventricular systolic pressure (LVSP) and the maximum change rate of left ventricular pressure rise and fall (±dp/dt) were significantly decreased (P<0.05, P＜0.01), while left ventricular end diastolic pressure (LVEDP) was significantly increased (P<0.05) in MI 48 h group.All the above indices in MI 4 weeks group had the same change as that in MI48h group, with the LVEDP significantly higher (P<0.01), except for a non-significantly change in SBP, DBP and MAP (all P>0.05).In both MI 48 h and MI 4 weeks groups, myocyte apoptotic index was significantly increased in the infracted/scar, border and non-infarcted areas (P<0.05,P＜0.01) with caspase-3 and Bax expressions increased significantly (P<0.05, P＜0.01) in myocytes of the above three areas and Bcl-2 expression increased only in myocytes of the infracted area in MI 48 h group.Western blotting indicated that Bcl-2/Bax ratio was also decreased in MI 48 h subgroup.CONCLUSIONS: After AMI in rats, cardiomyocyte apoptosis happened in the infarction/scar, border and non-infarcted areas, with caspase-3 and Bax expression in myocytes increased, and with Bcl-2 expression increased in myocytes of infracted area and Bcl-2/Bax ratio decreased only early after AMI.     

Adenosine A2A receptor antagonist SCH58261 attenuates hypoxic-ischemic brain damage in a fetal rabbit model

AIM: To study the effect of adenosine A_2A receptor antagonist SCH58261 on hypoxic-ischemic brain damage (HIBD) in a mature fetal rabbit model.METHODS: Pregnant New Zealand white rabbits at gestational day 29 were selected and were randomly divided into sham-operated group, hypoxic-ischemic group, SCH58261 0.04 mg/kg group, SCH58261 0.12 mg/kg group and DMSO group. The intrauterine rabbit HIBD model was established. All pregnant rabbits were subjected to cesarean section 24 h after the sham operation or experimental procedure to induce hypoxic-ischemic injury in the fetus. The survival neonatal rabbits were kept in a neonatal incubator at 35℃. The general conditions of the newborn rabbits were recorded. The degree of neurobehavioral damage in the newborn rabbits was estimated by a neurobehavioral scoring protocol. The concentration of SCH58261 in the serum of pregnant rabbits, the serum of neonatal rabbits and the brain tissues of neonatal rabbits was measured by mass spectrometry. The mRNA expression of Bcl-2/Bax and protein levels of p-P38 mitogen-activated protein kinase (MAPK) in the cortex, hippocampus and striatum area in the brain of the neonatal rabbits were determined by real-time PCR and Western blot. RESULTS: SCH58261 was detected in the serum and brain tissues of the newborn rabbits. The SCH58261 concentration was approximately 40 μg/L in the brain tissue of the newborn rabbits. The mRNA expression of Bcl-2 in the cortex, hippocampus and striatum of brain tissues in SCH58261 0.04 mg/kg group and SCH58261 0.12 mg/kg group was higher, and the mRNA expression of Bax was lower than those in HI group (P<0.05). The protein level of p-P38 MAPK in the cortex, hippocampus and striatum of brain tissues was reduced in SCH58261 0.04 mg/kg group and SCH58261 0.12 mg/kg group compared with HI group (P<0.05). The protein level of p-P38 MAPK in SCH58261 0.12 mg/kg group was a little lower than that in SCH58261 0.04 mg/kg group (P<0.05). CONCLUSION: Adenosine A_2A receptor antagonist SCH58261 attenuates hypoxia-ischemia induced neonatal brain injury by blocking adenosine A_2A receptor, subsequently inhibiting p-P38 MAPK phosphorylation to reduce neuronal apoptosis.     

Expression of genes in MAPKs pathway in development of neural tube defects induced by hyperthermia

AIM: To study the expression and the role of ERK1/2 and JNK1/2 of MAPKs pathways in the development of neural tube defects induced by hyperthermia. METHODS: The animal models of golden hamster were produced by hyperthermia. The expression of ERK1/2 and JNK1/2, and levels of their phosphorylation were measured by Western blotting in control group and hyperthermia group. RESULTS: p-ERK1/2 steadily expressed in each control group, and the expression of p-ERK1/2 significantly decreased, which was different from that in the corresponding control group (P<0.05). The activity of p-JNK1/2 increased in hyperthermia group and the amount of p-JNK1/2 increased as compared to control group. The peak appeared at 16 h after exposed to hyperthermia (P<0.05). CONCLUSION: Hyperthermia, which induces a decrease in p-ERK1/2 expression and increases the expression of p-JNK1/2 of MAPKs pathway, results in the unbalance of cell proliferation and apoptosis, and induces neural tube defects.     

Effects of isoflurane and sevoflurane on apoptosis of cortical neuron in neonatal rats and the role of MAPKs pathway

AIM: To investigate the effects of isoflurane and sevoflurane at the same dose on apoptosis of cortical neuron in neonatal rats and the role of mitogen-activated protein kinases (MAPKs) pathway.METHODS: Eleven neonatal rats were selected at postnatal day 7 from 1 litter (altogether 5 litters) and assigned randomly into control group (C group), isoflurane group (Ⅰ group) and sevoflurane group (S group). The rats in Ⅰ group, S group or C group were exposed to 1.1% isoflurane, 1.8% sevoflurane and room air for 4 h, respectively. The brain of neonatal rats were perfused and embedded by paraffin. Caspase-3 positive expression in the retrosplenial cortex (RS) of the brain was observed by immunohistochemical staining. Meanwhile, the fresh cortex was separated at 0 h in C group and at 2 h and 4 h in Ⅰ group and S group. The levels of phospho-SAPK/JNK and SAPK/JNK, phospho-p38 and p38 in fresh cortex were detected by Western blotting.RESULTS: Caspase-3 positive cells in the the cortex were increased by 441% in Ⅰ group (P<0.01) and 151% in S group (P<0.01) as compared to C group, and increased by 115% in Ⅰ group (P<0.05) as compared to S group. The protein levels of phospho-SAPK/JNK in the cortex were increased by 219% at 2 h (P<0.05) and 181% at 4 h (P<0.05) in Ⅰ group, while no significant difference between S group and C group was observed. The phospho-p38 protein in the cortex was increased by 38.9% at 2 h (P<0.05) and 36.9% at 4 h (P<0.05) in Ⅰ group, and increased by 32.6% (P<0.05) at 2 h and 128.0% at 4 h (P<0.01) in S group as compared to C group.CONCLUSION: Isoflurane induces more apoptotic neurons in the cortex of the brain in neonatal rats at postnatal day 7 than sevoflurane. Isoflurane induces apoptosis mainly by activating SAPK/JNK phosphorylation, while sevoflurane induces aopotosis by activating p38 phosphorylation.     

Lutein suppresses t-BHP-induced apoptosis in retinal ganglion cells

AIM: To investigate the protective effects of lutein on retinal ganglion cells in vitro. METHODS: The effect of lutein on tert-butyl hydroperoxide (t-BHP)-treated retinal ganglion cells (RGC-5 cell line) was determined. The protein expression of Brn-3 and MAP-2 was examined by the method of immunocytochemistry to identify the RGC-5 cells. The RGC-5 cells were induced by a 24 h exposure of t-BHP, and the cell viability was examined by MTT assay. The apoptotic ratio of the RGC-5 cells after exposed to t-BHP or/ and lutein treatment was analyzed by flow cytometry assay with Annexin V-FITC /PI staining. The activation of caspase-3 was detected by immunocytochemistry and the protein levels of Bcl-2, Bax, cleaved caspase-3, JNK and c-Jun were determined by Western blot. RESULTS: The RGC-5 cells expressed Brn-3 and MAP-2 proteins. Lutein treatment prevented t-BHP-induced RGC-5 cell apoptosis and increased the cell activity. Compared with control group, exposure of the RGC-5 cells to t-BHP decreased the expression of anti-apoptotic protein Bcl-2, increased the Bax/Bcl-2 ratio, up-regulated the level of cleaved caspase-3, also promoted the phosphorylation of JNK and c-Jun. Lutein partly reversed the effects of t-BHP on the RGC-5 cells mentioned above. CONCLUSION: Lutein protects RGC-5 cells against t-BHP-induced apoptosis by up-regulating Bcl-2 expression and inhibiting caspase-3 activation through modulating the JNK signaling pathway.     

Effects of maternal limb ischemic preconditioning on fetal hippocampal neuron apoptosis induced by intrauterine distress-reoxygenation in rats

AIM: To evaluate the influence of maternal limb ischemic preconditioning (LIP) on the apoptosis of fetal hippocampal neurons induced by intrauterine distress-reoxygenation in rats. METHODS: Intrauterine ischemia was induced by clamping the uterine and uterine branch of the ovarian blood vessels with aneurysm clamps for a period of 15 min followed by removal of the clamps to permit reperfusion. Sprague-Dawley (SD) rats (n=12) were randomly divided into 4 groups on the 19th pregnant day: sham (S) group, LIP group, fetal distress (FD) group and LIP+FD group. The cesarean birth occurred on embryonic day 21 to obtain 12 fetal rats alive in each group. The fetal rats were decapitated and the pyramidal cells in CA1 hippocampus were observed under light microscope. The neuronal apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and the apoptotic rate was calculated. The expression of Bcl-2 and Bax were detected by immunohistochemical method and Western blotting. RESULTS: The rates of neuronal apoptosis in FD group and LIP+FD group were significantly higher than that in S group (P<0.05), while no significant difference was observed between S group and LIP group (P>0.05). The expression of Bcl-2 and Bax in FD group and LIP+FD group was significantly higher than that in S group (P<0.05), while no significant difference was observed between S group and LIP group (P>0.05). The ratio of Bcl-2/Bax was lower in FD group than that in S group. Compared with FD group, the rate of neuronal apoptosis was significantly lower (P<0.05), while the ratio of Bcl-2/Bax was significantly higher (P<0.05) in LIP+FD group. CONCLUSION: Maternal limb ischemic preconditioning attenuates the apoptosis of fetal hippocampal neurons induced by intrauterine distress-reoxygenation in rats, which may be associated with the up-regulation of Bcl-2 expression.     

Neuronal apoptosis and the chance of apoptosis-related proteins in ovariectomied rats with the effect of App17-mer peptide

AIM: To observe the expression of apoptosis-related proteins in hippocampal neurons of ovariectomized (OVX) rats and explore the neuroprotective mechanism of the App17-mer peptide. METHODS: Female Wistar rats were randomly divided into three groups. Bilaterally ovariectomized rats with injection of App 17P peptide (3.5 μg in 0.1 mL/per rat, three times a week) formed the experimental group (17P+ OVX group). Anti-AIF, Bcl-2 and Bax antibodies were applied in the immunohistochemistry experiment. TUNEL was employed to detect apoptosis. RESULTS: The number of apoptotic neurons was clearly higher in hippocampal and cortex in OVX group than that in OVX+17P group. Immunohistochemistry demonstrated the increased expression of AIF, Bax in hippocampal neurons of OVX group. OVX group showed a significantly reduced expression of Bcl-2 in hippocampal neurons. Hippocampal tissue from OVX group showed the increased expression of AIF,Bax ,and showed diminished expression of Bcl-2 , treating with App17-mer peptide normalized the expression of these proteins. CONCLUSIONS: The expression of apoptosis-related proteins were abnormal in the OVX rats. App17-mer peptide normalized these changes ; Estrogen deficiency induced neuronal apoptosis. App17-mer peptide diminished apoptosis.     

Effects of celecoxib on cardiac myocyte apoptosis after myocardial infarction

AIM: To investigate the effects of celecoxib, a selective cyclooxygenase-2 inhibitor, on antioxidative capability and apoptosis of cardiac myocytes after myocardial infarction. METHODS: 24 New Zealand rabbits were divided into three groups randomly (8 in each group): sham-operated group (sham group), myocardial infarction group (MI group), celecoxib group (Cele group, 10 mg kg^-1·d^-1, qd, with the drugs gastric gavage for six weeks). The NO concentration, total antioxidative capability (T-AOC), the activity of constitutive nitric oxide synthase (cNOS) and inducible NOS (iNOS) in cardiac tissue homogenate, adjacent to the infracted area, were detected. The pathological changes were observed by light microscope and electron microscopy. The expressions of Bcl-2 and Bax protein in myocytes were observed using immunohistochemistry, and the degree of apoptosis were examined by TUNEL. RESULTS: Cardiac tissue in MI group presented interstitial edema, fibroplastic proliferation, inflammatory cellular infiltration, and vacuolar degeneration in cardiac myocytes. The results of electron microscopy showed that myocytes presented more changes caused by ischemic injury: widened interspace of myofibril, disordered myofibrillae, focal lysis of myofilament, ectasia of sarcoplasmic reticulum. In Cele group, the pathological changes were light, the NO^-₂/ NO^-₃ concentration, the activity of iNOS were lower (P<0.05), while the activity of cNOS and T-AOC were higher (P<0.05) than those in MI group. The expression rate of Bcl-2 protein in Cele group was higher than that in MI group, while the Bax was lower (P<0.01). The number of apoptotic myocytes was lower than that in MI group (P<0.01).CONCLUSION: Celecoxib decreases the number of apoptotic cardiomyocytes and increases the antioxidative capability after myocardial infarction.     

Schisandra total lignin attenuates apoptosis of endoplasmic reticulum pathway to delay mouse brain aging

AIM:To investigate the role of schisandra total lignin (SCL) in anti-aging of the mouse brain. METHODS:A D-galactose induced mouse aging model was established. The following groups in this study were set up: control group, model group, SCL low dose group ［SCL (L)］, SCL moderate dose group ［SCL (M)］ and SCL high dose group ［SCL (H)］. Learning and memory abilities were measured by mouse jumping experiments. The expression of ubiquitin (Ub), glucose-regulated protein 78 （GRP78）, protein disulfide isomerase (PDI), CCAAT /enhancer-binding protein homologous protein (CHOP), Bcl-2 and Bax proteins was detected by Western blotting. In addition, the protein expression of Bcl-2 and Bax in the aging cerebral cortex was also observed by immunohistochemistry. RESULTS:In learning test, compared with control group, the number of errors within 5 min increased in model group (P<0.05). Compared with model group, the number of errors within 5 min decreased in SCL (L) group, SCL(M) group and SCL(H) group (P<0.05). In memory test, compared with control group, incubation period of the first jumping off the platform was shorter and the number of errors within 5 min increased in model grou(P<0.05). Compared with model group, the incubation period of the first jumping off the platform was longer and the number of errors within 5 min decreased in SCL (L) group , SCL(M) group and SCL(H) group (P<0.05). Compared with control group, the protein expression of Ub, GRP78, PDI, CHOP and Bax was increased (P<0.05), while Bcl-2 protein level and Bcl-2/Bax ratio were decreased in model group(P<0.05). Compared with model group, the protein expression of Ub, GRP78, PDI, CHOP and Bax was decreased in SCL (L) group, SCL(M) group and SCL(H) group (P<0.05), while Bcl-2 protein level and Bcl-2/Bax ratio were increased (P<0.05). In control group, neuronal morphology was normal, the protein expression of Bcl-2 was positive and Bax was negative in the cytoplasm. In model group, the neurons were degeneration, the protein expression of Bcl-2 was negative and Bax was positive in the cytoplasm. In SCL (L) group, SCL (M) group and SCL (H) group, the number of degenerative neurons decreased, the protein expression of Bcl-2 was positive and Bax was negative in the cytoplasm. CONCLUSION: SCL inhibit D-galactose-induced brain aging in mice by attenuating apoptosis of endoplasmic reticulum pathway.     

All-trans retinoic acid attenuates blood-brain barrier injury induced by cerebral ischemia-reperfusion in rats through JNK/P38 MAPK signaling pathway

AIM: To investigate the effect of all-trans retinoic acid (ATRA) on blood-brain barrier after cerebral ischemia-reperfusion (CIR) injury in rats and its possible role mechanism.METHODS: Male SD rats were randomly divided into sham group, model (CIR) group and CIR+ATRA (10, 30 and 90 mg/kg) groups. The rat model of CIR injury was established by MCAO thread occlusion method. After ischemia for 1.5 h and reperfusion for 24 h, the neurological functional behavioral score, cerebral infarction volume, brain water content and Evans blue content were determined. The activity of matrix metalloprotein-9 (MMP-9) was measured by gelatin zymography. The protein levels of claudin-5, occludin, ZO-1, JNK, p-JNK, P38, p-P38 and MMP-9 in the brain tissues were determined by Western blot.RESULTS: Compared with CIR model group, ATRA at 30 mg/kg significantly improved neurological function, and decreased cerebral infarction volume, brain water content, Evans blue content and the degradation of tight junction proteins in ischemic area (P<0.01). The activity and protein expression of MMP-9 in ischemic brain tissue were decreased (P<0.01). The phosphorylation of JNK and P38 was inhibited and the protein levels of p-JNK and p-P38 were decreased (P<0.01).CONCLUSION: ATRA reduces the damage of brain tissue and the destruction of blood-brain barrier induced by CIR in rats. The protective effect may be related to inhibiting the activation of JNK/P38 MAPK signaling pathway and MMP-9.     

Effect of sodium ferulate on bcl-2 and bax gene expression of apoptotic hippocampal neurons induced by sodium ferulate

AIM: To investigate the protective effects of sodium ferulate (SF) on apoptosis in cultured hippocampal neurons induced by sodium nitroprusside (SNP), and the effect of SF on expression of bcl-2 and bax. METHODS: The primary cultured hippocampal neurons were exposed to 50 μmol SNP, a nitric oxide-donor, for 24 h after pretreatment with different concentrations of SF (10-160 μmol/mL) for 6 h. Then neuronal viability was tested by MTT assay. Fluorescent staining with Hoechst 33258 and agarose gel electrophoresis was used to analyze apoptosis. The expressions of bcl-2, bax mRNA and protein were tested by RT-PCR and Western blotting. RESULTS: Pretreatment with SF(10-160 μmol/L) for 6 h increased the survival rate of neurons. SF prevented the neuronal nuclei from shrinkage, condensation and cleavage and blocked neuronal nuclear DNA fragmentation induced by SNP. SF also increased the expressions of bcl-2 mRNA and Bcl-2 protein and decreased the expressions of bax mRNA and Bax protein. CONCLUSION: SF prevents the cultured hippocampal neurons against SNP neurotoxicity. The mechanism of protection is related to the increase in Bcl-2 level and the decrease in Bax level. As a result, the ratio of Bcl-2/Bax is changed.     

USP14 regulates H2O2 induced oxidative stress in H9c2 cells

AIM:To evaluate the effect of inhibiting ubiquitin-specific protease 14(USPl4) activity on oxidative stress induced by H₂O₂ of H9c2 cells.METHODS:The H9c2 cells were incubated with H₂O₂ at 25 μmol/L for 2 h to establish the oxidative stress injury model.The cells were divided into control group,H₂O₂ group,IU1 group (25 μmol/L or 50 μmol/L) and IU1+H₂O₂ group.The H9c2 cells activity was measured by MTS assay.The level of intracellular reactive oxygen species (ROS) and cell survival rate were analyzed by flow cytometry assay.The changes of the mitogen-activated protein kinase (MAPK) family related proteins were detected by Western blot.RESULTS:Compared with control group,the cell activity and the viability rate in H₂O₂ group were decreased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were increased (P<0.05).Compared with H₂O₂ group,the cell activity and the viability rate of the H9c2 cells in IU1+H₂O₂ group were increased (P<0.05),while the intracellular ROS,the protein levels of Bax/Bcl-2,P53,p-ERK1/2,p-JNK and p-P38 were decreased (P<0.05).CONCLUSION:Inhibition of USPl4 activity reduces the oxidative stress injury of the H9c2 cells.The mechanism may be related to inhibition of the MAPK signaling and down-regulation of apoptosis related proteins.     

Effect of JNK/MCP-1 signaling pathway on anti-diabetic neuropathic pain by curcumin in type 2 diabetic rats

AIM:To investigate the effect of JNK/MCP-1 signaling pathway on anti-diabetic neuropathic pain by curcumin in type 2 diabetic rats. METHODS:The male Sprague-Dawley rats were induced as the model of the type 2 diabetic neuropathic pain rats, they were randomly divided into 6 groups (n=27): type 2 diabetic neuropathic pain (DNP) group, type 2 diabetic neuropathic pain and intraperitoneal injection of curcumin (Cur) group, type 2 diabetic neuropathic pain and solvent control (DSC) group, type 2 diabetic neuropathic pain and JNK inhibitor (DJ) group, type 2 diabetic neuropathic pain and JNK inhibitor solvent control (DJS) group, type 2 diabetic neuropathic pain and monocyte chemoattractant protein 1 (MCP-1) agonist (DM) group. Another 27 normal SD rats were selected as control group. Mechanical withdrawal threshod and thermal withdrawal latency were measured at 3rd d, 7th d and 14th d after dosing, then the lumbar segment 4~6 of the spinal cord and L4~6 DRG were removed at the same time. ELISA was used to measure MCP-1 level. The expression of p-JNK was determined by Western blotting. RESULTS:Compared with DNP group, p-JNK was significantly decreased at 7th d and 14th d in Cur group, DJ group and DM group after treatment (P<0.05). Compared with C group, the MCP-1 was significantly declined in other 6 group after streptozotocin injection (P<0.05). Compared with DNP group, MCP-1 were significantly increased at 7th d and 14th d in Cur group and DJ group after treatment (P<0.05), and that in DM group was greatly decreased (P<0.05). CONCLUSION: The expression of p-JNK and MCP-1 was increased in DNP rats with spinal cord and dorsal root ganglion. The mechanism of curcumin reducing the neuropathic pain in type 2 diabetic rats might be through regulating the JNK/MCP-1 pathway.