Aim To investigate the effects of lentivirus mediated nerve growth factor ( NGF) gene silencing on pheochromocytoma cells ( PC12 ) and the possible mechanisms .Methods The NGF shRNA expression vector was constructed .PC12 cells were randomly divi-ded into five groups (n=3 each) as follows: negative control group ( NC ) , control lentivirus group ( LV CON) , lentivirus NGF shRNA1 group ( LV shNGF1 ) , lentivirus NGF shRNA2 group(LV shNGF2), lentivir-us NGF shRNA3 group(LV shNGF3).The cells in NC group were cultured in DMEM/HG and polybrene me-dium, while others were cultured in DMEM/HG, poly-brene and corresponding lentivirus medium .After the treatment, the infection efficiency was determined by fluorescent microscope .Relative expression of NGF , extracellular signal-regulated kinase ( ERK1/2 ) and p-ERK1/2 were assessed by Western blot .The expres-sion of NGF mRNA was analyzed by quantitative re-verse transcription polymerase chain reaction ( qRT-PCR) .The differentiation degree was valued according to the length of neuritis and max diameter of cells .The cell viability was detected by CCK-8.Results The in-fection efficiency in PC12 cells reached over 90%. Compared with NC group , the relative expression of NGF mRNA and NGF protein was significantly down-regulated ( P<0.05 ) .There was no difference in the expression of ERK1/2 protein and cell viability .The expression of p-ERK1/2 protein was markedly down-regulated in LV shNGF3 group ( P<0.01 ) .The cells morphology was changed , and the length of neuritis and max diameter of cells were strained in LV shNGF 3 group than those in NC group ( P<0.01 ) .Conclusion Lentivirus-mediated NGF gene silencing inhibits the differentiation of PC12 cells through suppressing the activation of ERK1/2.

Aim To investigate the role of NO/cGMP in the cardioprotective effects of intrathecal morphine preconditioning against myocardial ischemia-reperfu-sion injury in rats. Method 54 Male Sprague-Dawley Rats were used to establish the model of intrathecal catheter placement. The rats were randomly assigned to
9 groups. SHAM (sham group), CON (control, sa-line) , ITMP ( intrathecal morphine preconditioning, 3μg·kg-1 ) , L-NAME+ITMP ( NO synthetase inhibi-tor,L-NAME ) , ODQ + ITMP ( guanylate cyclase in-hibitor, ODQ ) , KT5823 + ITMP ( PKG inhibitor, KT5823),L-NAME,ODQ,KT5823,6ratsineach
group. ITMP were produced by three cycles of 5 min intrathecal injection of morphine and 5 min intermis-sion before myocardial ischemia, CON were achieved by intrathecal injection of saline in the same way, L-NAME+ITMP, ODQ +ITMP, KT5823 +ITMP were prepared by intrathecally administering L-NAME ( 30 nmol), ODQ(11 nmol) and KT5823(20 pmol) 10 minutes prior to ITMP respectively, L-NAME, ODQ, KT5823 worked as the control of inhibitors themselves respectively without ITMP. Subsequently, all rats were subjected to 30 min of left coronary artery occlusion followed by 2 h of reperfusion except the SHAM group. Myocardial infarct size, as a percentage of the AAR,
was determined by 2 , 3 , 5-triphenyltetrazolium stai-ning. Results Compared with CON, the volumes of IS and IS/AAR were reduced in ITMP ( P <0.01 );the protective effects of ITMP were abolished by pre-treatment with L-NAME, ODQ and KT5823 ( P <0.01 );Conclusions NO/cGMP might be involved in the cardioprotective effect of intrathecal morphine pre-conditioning against myocardial ischemia and reperfu-sion injury in rats.

Objective To explore the effect of hyperbaric oxygen (HBO) preconditioning on learning and memory damage induced by high positive acceleration( + Gz) exposure in rats.Methods Thirty-two male Sprague-Dawley rats were randomly divided into four groups with 8 in each group: control group( Con), + Gz group,HBO group and HBO-+ Gz group.Rats of Con group were given 5d( 1 ATA ,21% O2, 1h/d); Rats of + Gz group was exposed to + 10Gz for 5 min; HBO group were only given 5d (2.5 ATA, 100% O2,1 h/d); HBO-+ Gz group were given HBO 5 consecutive days,and then suffered +Gz exposure.Morris water maze was used to observe the navigation and probe capabilities of rats.Results In the spatial acquisition test,there exist significant difference among these groups(F(3.28) = 5.325, P＜ 0.01 ).Compared with the control group, the escape latency increased significantly in the + Gz group and HBO-+ Gz group (P＜0.05) while had no difference in HBO group.HBO-+ Gz group had significantly shorter escape latency than + Gz group (P＜0.05).In the probe test,compared with the control group, + Gz group and HBO-+ Gz group had a longer percentage in the target quadrant( (43.71 ± 3.29 ) %vs (28.65 ±1.00)%, P＜0.05;(43.71 ±3.29)% vs (37.17 ±0.98)%, P＜0.05)),and HBO-+Gz group was better than + Gz group.Conclusion HBO preconditioning may have a protective effect on the impairment of learning and memory caused by + Gz exposure in rats.

Objective To investigate the effect of intrathecal morphine preconditioning on the expression of nerve growth factor (NGF) in the dorsal root ganglia (DRG) in a rat model of myocardial ischemia-reperfusion (I/R).Methods Thirty healthy adult male Sprague-Dawley rats in which intrathecal catheters were successfully placed without complications,weighing 250-350 g,were randomly divided into 5 groups (n =6 each) using a random number table:sham operation group (S group),I/R group,intrathecal morphine preconditioning group (ITMP group),μ receptor antagonist CTOP + intrathecal morphine preconditioning group (CTOP + ITMP group),and CTOP control group (CTOP group).Myocardial ischemia was induced by 30 min of occlusion of the anterior descending branch of the left coronary artery followed by 120 min of reperfusion in all the groups except S group.Intrathecal morphine preconditioning was produced by 3 cycles of 5 min intrathecal injection of morphine 3 μg/kg (10 μl) at 5 min intervals within 30 min before ischemia in ITMP group.In CTOP+ITMP and CTOP groups,1 μg/μ1 CTOP 10 μl was injected intrathecally at 10 min before morphine preconditioning and 40 min before ischemia,respectively.At 120 min of reperfusion,the rats were sacrificed,and myocardial specimens were obtained for determination of myocardial infarct size,and DRGs were removed for determination of the expression of NGF by using immunohistochemistry and Western blot.Results Compared with S group,the myocardial infarct size was significantly increased,and the expression of NGF in DRGs was significantly up-regulated in I/R group (P＜0.05).Compared with I/R group,the myocardial infarct size was significantly decreased,and the expression of NGF in DRGs was significantly down-regulated in ITMP group (P＜ 0.05),and no significant change was found in the parameters mentioned above in CTOP group (P＞0.05).Compared with ITMP group,the myocardial infarct size was significantly increased,and the expression of NGF in DRGs was significantly up-regulated in CTOP+ITMP and CTOP groups (P＜0.05).Conclusion The mechanism by which intrathecal morphine preconditioning reduces myocardial I/R injury is related to activation of spinal μ receptors,inhibition of NGF expression in DRGs,and reduction of responses to noxious stimulation in the rats.

Objective To investigate the role of p38 mitogen-activated protein kinase (p38MAPK) signaling pathway in reduction of myocardial ischemia-reperfusion (I/R) injury by morphine preconditioning in the rats with chronic heart failure in vitro.Methods Adult male Sprague-Dawley rats,weighing 200-230 g,aged 6-7 weeks,in which doxorubicin 2 mg/kg was injected via the tail vein once a week for 6 consecutive weeks to induce chronic heart failure,were studied.At the end of 8th week,30 rats with chronic heart failure were randomly divided into 5 groups (n =6 each) using a random number table:sham operation group (group Sham),I/R group,morphine preconditioning group (group MPC),SB203580 (p38MAPK inhibitor) + morphine preconditioning group (group SBM),and SB203580 group (group SB).The hearts were quickly excised and passively perfused in a Langendorff apparatus and subjected to 30 min of occlusion of the anterior descending branch of the left coronary artery followed by 120 min of reperfusion to establish the model of myocardial I/R injury.After equilibration,the hearts were subjected to 3 cycles of 5 min perfusion with K-H solution containing morphine 1 μmol/L at 5-min intervals before ischemia in group MPC.In group SBM,the hearts were perfused with K-H solution containing SB203580 (5 μmol/L) for 45 min starting from l0 min before morphine preconditioning until 5 min of ischemia.In group SB,morphine preconditioning was not performed,and the hearts were only perfused with K-H solution containing SB203580 (5 μmol/L) starting from 40 min before ischemia until 5 min of ischemia.At 15 min of equilibration (baseline),5 and 10 min of reperfusion,the coronary effluent was collected to detect the activity of lactate dehydrogenase (LDH) using the chemical colorimetry.At 10 min of reperfusion,the expression of phosphor-p38MAPK (p-p38MAPK) in the myocardium was determined by Western blot in Sham,I/R and MPC groups.At 120 min of reperfusion,the area at risk (AAR),total areas of right and left ventricles (LV+RV),and infarct size (IS) were measured,and the IS/AAR ratio was calculated.Results Compared with group Sham,the LDH activity in coronary effluent during reperfusion and IS/AAR ratio were significantly increased in the other groups,and the expression of p-p38MAPK was significantly up-regulated in I/R and MPC groups (P＜0.05).Compared with group I/R,the LDH activity in coronary effluent during reperfusion was significantly decreased,the expression of p-p38MAPK was significantly up-regulated,and the IS and IS/AAR ratio were significantly decreased in group MPC (P＜0.05),and no significant change was found in the LDH activity in coronary effluent,IS and IS/AAR ratio in SBM and SB groups (P＞0.05).Compared with group MPC,the LDH activity in coronary effluent during reperfusion was significantly increased,and the IS and IS/AAR ratio were significantly increased in group SBM (P＜0.05).Conclusion The mechanism by which morphine preconditioning reduces myocardial I/R injury is related to activation of p38MAPK signaling pathway in the rats with chronic heart failure in vitro.

Objective To investigate microRNAs ( miRNAs) expression profiling of cardiomyocytes in rats with heart failure, and predict miRNAs-regulated target genes and their functions.Methods Total of 18 male SD rats weighing 200-220 g were randomly divided into 2 groups:the control group ( CON) and the heart failure group (HF).The rats in HF group were injected by adriamycin via tail vein to induce heart failure, meanwhile in CON group, rats were received an equal volume of 0.9% sodium chloride intravenously.The cardiomyocytes isolated from the rat hearts in two groups and cultured overnight.After that, total RNA was extracted and then subjected to miRNA microarray to screen differentially expressed miRNAs.The reults of microarray were further verified by quantitative real-time PCR ( qRT-PCR ) .The target genes regulated by differentially expressed miRNAs were predicted by the software of Targetscan and miRanda.Bioinformatics analysis was performed to predict the miRNAs-regulated target genes and analyze the enriched gene ontology ( GO) and signaling pathway ( KEGG Pathway) .Results The results of miRNA microarray showed that a total of 37 miRNAs were differentially expressed in HF group as compared to CON group, among which 22 miRNAs were up-regulated and 15 miRNAs were down-regulated (P<0.01, FDR<0.05).The expression of miR-133b-5p (t=14.56, P<0.01), miR-6216 (t=9.32, P<0.01) and let-7e-5p (t=13.92, P<0.01) which were detected by qRT-PCR exhibited the similar tendency of up or down regulation to those shown in microarray results.Bioinformatics analysis indicated that miRNAs-regulated target genes were significantly enriched in 31 GOs (P<0.01, FDR<0.05) and 12 signal pathways (P<0.05, FDR<0.05), among which ubiquitin-proteasome system, MAPK signaling pathway and Toll like siganling pathway exhibited a higher enrichment. Conclusion MiRNA expression profile on cardiomyocytes in rat with adriamycin-induced heart failure was significantly changed.These differentially expressed miRNAs might participate in the process of heart failing by regulating their target genes in rat cardiomyocytes.

Objective To investigate the effect of intrathecal morphine preconditioning (ITMP) on the excitability of substantia gelatinosa (SG) neurons in the dorsal horn of the spinal cord in a rat model of myocardial ischemia-reperfusion (I/R).Methods Thirty-six adult male Sprague-Dawley rats,weighing 200-300 g,in which intrathecal catheters were successfully placed without complications,were randomly divided into 3 groups (n =12 each) using a random number table:sham operation group (group S),group I/R,and group ITMP.Myocardial I/R injury was produced by occlusion of the left anterior descending branch of the coronary artery for 30 min followed by 120 min reperfusion.In group ITMP,the rats received intrathecal morphine 3 μg/kg (10 μl) by three cycles of 5 min infusions interspersed with 5 min infusion-free periods starting from 30 min before ischemia,and the equal volume of normal saline was given instead of morphine in group I/R.At 10 min of reperfusion,6 rats randomly selected in each group were sacrificed,and the T2-6 segments of the spinal cords were acutely isolated to prepare spinal cord slices.The resting potential,threshold of action potential (APT),peak of action potential (APP) and action potential duration in SG neurons in the dorsal horn of spinal cord slices were determined using the whole-cell patch-clamp technique,and the number of action potentials evoked by currents of 40,60,80 and 100 pA was recorded.At 120 min of reperfusion,6 rats randomly selected in each group were sacrificed,and myocardial specimens were obtained for determination of myocardial infarct size (IS) and area at risk (AAR),and IS/AAR ratio was calculated.The expression of c-fos in the T2-5 dorsal horns of the spinal cords was detected by Western blot.Results Compared with group S,the IS/AAR ratio was significantly increased,the expression of c-fos was up-regulated,the number of action potentials in SG neurons in dorsal horns of spinal cord was increased,APT was decreased,and APP was increased in group I/R (P＜0.05).Compared with group I/R,the IS/AAR ratio was significantly decreased,the expression of c-fos was down-regulated,the number of action potentials in SG neurons in dorsal horns of spinal cord was decreased,APT was increased,and APP was decreased in group ITMP (P＜0.05).Conclusion The mechanism by which ITMP attenuates myocardial I/R injury is related to decrease in the excitability of SG neurons in the dorsal horn of the spinal cord and reduction of responses to nociceptive stimuli in rats.

Objective evaluate the role of vagus nerve?muscarinic cholinergic receptor ( M recep?tor) pathway in mitigation of myocardial ischemia?reperfusion (I∕R) injury by intrathecal morphine postcon?ditioning in rats. Methods Seventy adult male Sprague?Dawley rats in which intrathecal catheters were suc?cessfully placed without complications, weighing 250-350 g, were randomly assigned into 7 groups ( n=10 each) using a random number table:sham operation group (Sham group), I∕R group, intrathecal morphine postconditioning group ( MP group) , vagal transection ( VT) group, VT+ intrathecal morphine postcondi?tioning group (VT+MP group), atropine (ATP, M receptor antagonist) + morphine postconditioning group ( ATP+MP group) , and ATP group. Myocardial I∕R was produced by occlusion of the anterior descending branch of left coronary artery for 30 min followed by 2 h of reperfusion. Morphine ( 3μg∕kg, 10μl) was in?trathecally infused over 5 min starting from onset of reperfusion in MP group. Normal saline 10 μl was in?trathecally infused over 5 min starting from onset of reperfusion in NS group. The bilateral vagus nerves were transected at 10 min before reperfusion in VT+MP group. Atropine ( 0?1 mg∕kg, 0?5 ml) was intravenously infused over 5 min starting from 10 min before reperfusion in ATP+MP group. The occurrence of cardiac ar? rhythmia ( premature ventricular contractions ( PVCs) and ventricular tachycardia ( VT)∕ventricular fibrilla?tion ( VF) ) within the first 30 min of reperfusion was recorded. The rats were sacrificed at 120 min of reper?fusion, and myocardial specimens were obtained for determination of myocardial infarct size ( IS) as a per?centage of area at risk (AAR). IS∕AAR ratio was calculated. Results Compared with Sham group, the number of PVCs and VT∕VF and IS∕AAR ratio were significantly increased in the other groups. Compared with I∕R group, the number of PVCs and VT∕VF and IS∕AAR ratio were significantly decreased in MP group. Compared with MP group, the number of PVCs and VT∕VF and IS∕AAR ratio were significantly in?creased in VT+MP and ATP+MP groups. Conclusion Vagus nerve?M receptor pathway is involved in miti?gation of myocardial I∕R injury by intrathecal morphine postconditioning in rats.

Objective To investigate the parental origin for a rare case of complete hydatidiform mole and coexisting fetus and to discuss its diagnosis and differential diagnosis.Methods Tissues from the fetus,mole and placenta were collected and pathology analysis and chromosome analysis were done.The DNA from the fetus,mole and parents' peripheral blood leukocytes was amplified with five short tandem repeat (STR) markers (D4S2460,D18S488,D21S2039,DXS1205 and DYS219) at the same time to confirm the parental source of the hydatidiform.Results (1) Casereport:A 27-year-old woman,gravida 1,para 0,was found high risk for neural tube defects at 20 weeks of gestation.At 24+5 weeks of gestation,ultrasound examination demonstrated a normal fetus,a normal placenta and a huge mass with a multicystic appearance attached to the placenta with an obvious demarcation.The fetus died at 26 weeks of gestation.Serum human chorionic gonadotropin-β(β -hCG) level decreased obviously during the first two weeks after artificial induction,but elevated at the third week,and β-hCG titers fell to normal after 2 courses of chemotherapy.Fetus autopsy showed no structure abnormality.Histopathologic examination of the hydatidiform showed swelling of chorionic villi with hyperplasia of the trophoblast and formation of central cisterns suggesting of a twin pregnancy consisting of a complete hydatidiform mole and coexisting fetus.(2) Genetic analysis:The karyotype analysis of the normal placental villi was 46,XY; the cell cultures of fetal cartilage tissue and hydatidiform were failed.STR analysis showed that the fetus was diploid from biparental source;the mole was androgenetic source.And the mole had locus both from Y and X chromosome of the father,so it was heterozygous.It was suggested that this case was derived from one single oocyte fertilized with three spermatozoas.Conclusions STR analysis could be used to confirm the diagnosis of complete hydatidiform mole and coexisting fetus and to find the pathogenetic rnechanism.

Objective To evaluate the effect of sevoflurane postconditioning on inositol-requiring enzyme 1 (IRE1) signaling pathway in the brain tissues in a rat model of hemorrhagic shock and resuscitation (HSR).Methods Sixty healthy adult male Sprague-Dawley rats,weighing 300-350 g,were divided into 5 groups (n =12 each) using a random number table:sham operation group (group Sham),group HSR,1.2％ sevoflurane postconditioning group (group SP1),2.4％ sevoflurane postconditioning group (group SP2) and 3.6％ sevoflurane postconditioning group (group SP3).Hemorrhagic shock was induced by withdrawing blood (40％ of the total blood volume) from the right common carotid artery over an interval of 30 min,and 1 h later the animals were resuscitated with the shed blood infused via the left jugular vein over 30 min.SP1,SP2 and SP3 groups inhaled 1.2％,2.4％ and 3.6％ sevoflurane,respectively,for 30 min starting from the beginning of infusion of the shed blood.Oxygen was inhaled for 30 min instead of sevoflurane in Sham and HSR groups.Mean arterial pressure was recorded before withdrawing blood (T0),immediately after the end of withdrawing blood (T1),at 30 min after the end of withdrawing blood (T2),before infusion of the shed blood (T3),and immediately after infusion of the shed blood (T4).Arterial blood samples were obtained at T0,T1,T3 and T4 for blood gas analysis.Morris water maze test was performed at 72 h after the end of infusion of the shed blood.The animals were then sacrificed,and brains were removed for determination of the expression of caspase-3 in hippocampal CA1 region (by immunohistochemistry) and expression of IRE1 and X-box binding protein 1 (XBP1) in hippocampal tissues (by Western blot).Results Compared with group Sham,mean arterial pressure was significantly decreased at T1-3,the pH value and base excess were decreased,lactic acid concentrations were increased,the escape latency was prolonged,the frequency of crossing the original platform was decreased,and the expression of caspase-3 in hippocampal CA1 regitn and IRE1 and X BP 1 in hippocampal tissues was up-reg ulated in group HSR (P＜0.05).Compared with group HSR,the escape latency was significantly shortened,the frequency of crossing the original platform was increased,and the expression of caspase-3 in hippocampal CA1 region and IRE1 and XBP1 in hippocampal tissues was down-regulated in SP2 and SP3 groups (P＜0.05),and no significant changes were found in the parameters mentioned above in group SP1 (P＞0.05).Conclusion The mechanism by which sevoflurane postconditioning reduces brain injury may be related to activating IRE1 signaling pathway in the brain tissues in a rat model of HSR.