Background and purpose: AMP-activated protein kinase (AMPK) plays an important role in the regulation of cell metabolism and energy balance and is associated with cell proliferation, survival and multiple signaling pathways. Recent reports found that AMPK is involved in tumor suppression and drug resistance. The aim of this study was to explore the effect of AMPK on the anti-tumor effect of adriamycin and underlying mechanism in breast cancer MCF-7/adr cells. Methods:The anti-proliferative effects of adriamycin was detected by methyl thiazolyl tetrazolium (MTT) assay in MCF-7/adr, MCF-7/adr-vector and MCF-7/adr-AMPKαcells. The cell morphology in each group was stained with the lfuorescent dye Hoechst 33528, and the effects on apoptosis induction were examined by lfow cytometry (FCM). The intracellular concentration of adriamycin was detected by lfuorescence assay. The resis-tance-and apoptosis-related proteins were analyzed by Western blot. Results:The growth of breast cancer MCF-7/adr cells was inhibited by adriamycin in a dose-and time-dependent manner. The IC50 values at 24 and 48 h were (36.8±2.1) and (28.8±1.3) μg/mL, respectively. AMPKαover-expression enhanced the cytotoxic effect of adriamycin in MCF-7/adr-AMPKαcells in a dose-and time-dependent manner. Its IC50 values at 24 and 48 h were (16.0±0.7) and (4.2±0.2) μg/mL, respectively. Fluorescent morphological assay showed that AMPKαoverexpression contributed to adriamycin induced apoptosis in MCF-7/adr-AMPKαcells. After treatment with 1.0 μg/mL adriamycin for 48 h, the apoptosis rates of MCF-7/adr, MCF-7/adr-vector and MCF-7/adr-AMPKα cells were (12.0±1.4)%, (12.7±1.6)% and (32.0±4.2)%, respectively, indicating that overexpression of AMPKα enhanced the adriamycin-induced apoptosis in MCF-7/adr cells. Fluorescence microplate assay showed that over expression of AMPKαsigniifcantly increased the intracellular accumulation of adriamycin, in a concentration dependent manner. Western blot analysis showed that, compared with MCF-7/adr and MCF-7/adr-vector cells, the expressions of Bax, caspase-3 and cleaved PARP proteins were increased. Meanwhile, Bcl-2 and P-gp protein expressions were decreased in MCF-7/adr-AMPKαcells. Furthermore, the release of cytochrome c from mitochondria into the cytosol was also observed in MCF-7/adr-AMPKαcells. Conclusion:AMP-Kαoverexpression can enhance the chemosensitivity of breast cancer MCF-7/adr cells to adriamycin through inhibiting the drug effux transporter and regulating the expression of apoptosis-related proteins.

ObjectiveTo investigate the expression of paired immunoglobulin-like receptor B (PirB) in optic nerve, visual cortex, cerebella, spinal cord and sciatic nerve of normal adult mice.MethodsTwelve healthy adult BALB/c mice were randomly and equally divided into two groups.The immunohistochemistry and Western blot were used to detect the expression of PirB in the tissues described above respectively.ResultsBoth the immunohistochemistry and Western blot test revealed that the expression of PirB was positive in the optic nerve, visual cortex, cerebella and spinal cord, but negative in the sciatic nerve.The positive signals in the sections were located in the cell bodies and the neurites were observed in some of them.Western blot showed the apparent positive band of PirB in the optic nerve, visual cortex, cerebella and spinal cord rather than in the sciatic nerve.The protein expression level of PirB was relatively high in the visual cortex (P ＜0.05) but relatively low in the optic nerves (P ＜0.01).ConclusionThe PirB expresses positively in the optic nerve, indicating that PirB protein may closely correlate with the poor regeneration of the optic nerve.

Background Retinal ganglion cell (RGCs) death following ischaemic insult is the major cause of a number of vision-threatening diseases.Recent studies confirmed that micro RNA (miR-30b) can alleviate hypoxy-induced cardiac injury.However,whether miR-30b can protect RGCs against oxygen-glucose deprivation damage is still not ellucidated.Objective The aim of this study was to investigate the protective effect of miR-30b on RGCs damage caused by oxygen-glucose deprivation.Methods The retinas were isolated from the eyeballs of eight SD rats aged postnatal 24 hours and RGCs were primarily cultured.The cells were divided into the recombinant adeno-associated virus (rAVV) control group,rAAV-miR-30b mimic group and AAV-miR-30b inhibitor group.Then the cells were transfected using rAVV-miR plasmid,rAAV-miR-30b mimic plasmid and AAV-miR-30b inhibitor plasmid,respectively for 6 days with the RGCs ∶ AAV as 1 ∶ 10 000.The cells were cultured with low glucose medium in hypoxygen incubator (5％ CO2,17％ N2,3％ O2) or 5％ CO2 incubator respectively for 24 hours.Cell viability was detected by cell counting kit-8 assay.The expression of Tubulin Ⅲ,a neuron specific marker,was detected by immunofluorescence technology to evaluate the survival of RGCs.The apoptosis and necrosis of the cells were assessed by Hoechst/PI double staining.Results The RGCs grew well with round shape and 1 3 processes 7 days after cultured in the normal cells.However,the RGCs were diminished and the cell process disrupted in the oxygen-glucose deprivation group.The relative vability of the cells was 3.310-±0.162 in the rAAV-miR-30b mimic group,which was significantly higher than 0.949±0.141 in the rAAV-miR-30b inhibitor group and 0.900±0.181 in the rAAV-miR control group(t=10.508,10.296,both at P＜0.001).It was positively expressed in survival RGCs,with the red fluorescence.The number of Tubulin Ⅲ+ cells was (13.800± 1.924)/field in the rAAV-miR-30b mimic group,showing a significant increase in comparison with (0.600±0.548)/field in the rAAV-miR-30b inhibitor group and (0.800± 1.304)/field in the rAAV-miR control group (t =15.141,14.912,both at P ＜ 0.001).Significant differences were found in the apoptosis rate and necrosis rate among the rAAV-miR-30b mimic group,rAAV-miR control group and PBS group (F=10.851,P=0.002;F=6.378,P=0.013),and the apoptosis rate and necrosis rate in the rAAV-miR-30b mimic group were considerably lower than those in the rAAV-miR control group and PBS group (all at P＜0.05).Conclusions The oxygen-glucose deprivation models can be established in RGCs by hypooxygic and low-glucose cultivation.rAAV encoding miR-30b mimics transfection can protect RGCs against oxygen-glucose deprivation damage.

AIM To investigate the tissue distribution of exendin-4 after administration in healthy rats. METHODS Exendin-4 was radioiodinated by the Iodo-GenTMmethod. Tissue distribution of [125I]exendin-4 was investigated after sc administration of [125I]exendin-4 at 3 μg·kg-1 in rats. Both total radioactivity and trichloroacetic acid (TCA) precipitated radioactivity were used to calculate the levels of [125I]exendin-4 in rats plasma and tissue samples after sc administration. RESULTS The tissue distribution of [125I]exendin-4 after sc injection showed substantial disposition in kidneys, lungs, bladder and pancreas. The rank order of normalized tissue distribution was kidneys＞lungs＞bladder＞pancreas＞intestine＞plasma＞adrenals>jejunum＞lymph＞liver＞spleen＞heart＞marrow＞thymus＞testicles＞brain＞muscle＞adipose. CONCLUSION [125I]Exendin-4 underwent a rapid and wide distribution in the tissues throughout the whole body within the time course examined. TCA precipitated radioactivity in kidneys was the highest, however, only trace amounts of [125I]exendin-4 was detected in the brain.

Objective To investigate the clinical value of endoscopy in the treatment of choledocholithiasis. Methods All 227 patients underwent endoscopic treatment. 14 of them with stones less than 1. 0 cm in diameter were treated with endoscopic papillary balloon dilation; 194 patients with stones 1. 0-1. 5 cm in diameter were treated with endoscopic sphincterotomy; and 19 patients with stones up to 1.5 cm in diameter were treated with endoscopic mechanical lithotripsy. Results Choledocholithiasis were not confirmed by choledochography in 34 cases ( 15% , 34/227) , who underwent exploration of common bile duct through EPBD or EST, but no stones were found. In 187 of the 193 choledocholithiasis patients their stones were removed, the overall success rate with complete stones clearance was 96.9% ( 187/193). The incidence of complication was totally 5. 29% ( 12/227) , including acute cholangitis in 3 patients, acute pancreatitis in 8 patients, and bleeding in 1 patient. Conclusions Endoscopic treatment should be the first choice of patient with choledocholithiasis due to its advantages of safety, effectiveness and with less complication. EPBD or EST was not recommended in case of the common bile duct stones were not confirmed by ERCP, in order to avoid the risk of papilla injury.

Objective To observe the variation of dynamic 64-slice CT perfusion imaging of rats with traumatic brain injury and discuss the relating pathophysiological basis.Methods A total of 80 adult male SD rats were randomly divided into three groups according to random number table,ie,normal control group,sham injury group and injury group.The injury group was divided into eight subgroups at time points of 2,6,12,24,48,72,120 and 168 hours.The detection of CT perfusion imaging,water content and blood-brain barrier permeability was done in the injured rats at all time points.The pathological changes were also observed to calculate their correlation with CT perfusion parameters of the injured region.Results The relative value of the blood perfusion was decreased significantly to the mimimum within 24 hours after injury.Within 2-12 hours,relative cerebral fluid(rCBF)and relative cerebral blood volume(rCBV)remained in a low perfusion state,with just a little increase.Relative mean transit time(rMTT)was prolonged and permeability surface(PS)increased.rCBF and rCBV were increased gradually with time,which was reversed till at 24 hours after injury and the injured side was in a high perfusion state,with the highest value of PS.The perfusion reached peak at 48 hours after injury and then became normal gradually.The water content was increased at 2 hours after injury and reached its peak at 48 hours.The permeability of blood-brain barrier(BBB)began to increase at 2 hours after injury and reached the peak at 24 hours.rCBF and rCBV were positively correlated with change of brain edema and PS was positively correlated with BBB permeability.Conclusion The dynamic 64-slice spiral CT perfusion imaging reflects the variation of BBB and edema and can be used as noninvasive imaging method for predicting the degree of brain perfusion and edema.

Objective To investigate the protecting mechanism of heat stress pretreatment on acute lung injury(ALI). Methods The oleic acid ALI mouse model was built to dynamically observe the binding capacity and the binding affinity of glucocorticoid receptor(GR),the levels of GR,heat shock protein 90(Hsp90)and Hsp70 before and after hyperthermic stress pretreatment. Results Heat stress pretreatment had significant protective effect on ALI.Western blotting showed insignificant changes of GR levels but progressive increase of level of Hsp70 and Hsp90.Heat stress pretreatment exerted insignificant effect on Bmax and Kd of GR,shown by radio ligand binding assay after ALI. Conclusion The protective effects of heat stress pretreatment on ALI of mouse may relate to its ability of keeping stable GR level and increasing levels of Hsp70 and Hsp90.

Objective To explore the role of H19 imprinting in etiology of pre-eclampsia. Methods Placentas of 24 women with pre-eclampsia (3 with mild pre-eclampsia and 21 with severe pre-eclampsia) and 50 healthy pregnant women at full term (control) were collected during selected cesarean delivery between August 2007 and March 2008. The statuses of H19 imprinting with placental tissues from normal pregnancy and patients with pre-eclampsia were identified upon polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The systolic and diastolic pressure were analyzed in H19 heterozygotic women. Results (1) There were 20 (40%) heterozygotes in 50 cases placenta tissues of the third trimesters, 11 (45%) heterozygotes in 24 cases placenta tissues of pre-eclampsia, There were no significant difference between two groups ( P > 0.05 ). (2) All 20 heterozygotes in placenta tissues of the third trimesters are exclusively monoallelically expressed, while 5 cases (45%) in 11 heterozygotes of pre-eclampsia are biallelically expressed (loss of imprinting, LOI). There were significant difference between two groups (P < 0. 01 ). (3) The values of systolic and diastolic pressure of patients with monoallelic expression of H19 were (171 ±9) mm Hg (1 nun Hg =0.133 kPa) and ( 104±8) mm Hg, the values of systolic and diastolic pressure with biallelic expression were ( 194±21 ) mm Hg and ( 124±18) mm Hg. There were significant difference between two groups (P<0.05 ). Conclusion LOI of H19 can be identified in pre-eclamptic placentas and is associated with maternal blood pressures, which implies the involvement of H19 gene LOI in the pathogenesis of pre-eclampsia and its potential relationship with the severity of the disease.

Objective To investigate the effect of adenosine A2A receptor on pituitary-adrenal axis response in acute phase of moderate craniocerebral trauma.Methods Eighteen adenosine A2A receptor knock-out mice in a C57BL/6 background and another eighteen their wild-type littermates were divided into normal control group and craniocerebral trauma for 4 hours group,and craniocerebral trauma for 24 hours group according to random number table,with siμ mice per group.Plasma levels of adrenocorticotropic-hormone (ACTH) and corticosterone at hours 4 and 24 postinjury were determined using ELISA method.Results At 4 and 24 hours,brain water content in wild-type mice [(80.950 ± 0.184) ％,(82.178 ± 0.255)％ respectively] was higher than that in gene knock-out mice [(80.006 ± 0.199)％,(81.091 ± 0.295)％ respectively,P ＜ 0.01].Besides,brain water content in both wild-type and gene knock-out mice increased after injury (P ＜ 0.01).Plasma levels of ACTH and corticosterone were higher in geneknock-out sham mice than in wild-type sham mice [(120.214 ± 2.472) ng/L vs (91.767 ±7.395) ng/L,(27.814 ±0.888) μg/L vs (11.430 ±0.644) μg/L respectively,P ＜0.0l].At 4 and 24 hours,plasma levels of ACTH [(174.776-± 5.040) ng/L,(189.613 ± 4.802) ng/L respectively] in geneknock-out mice showed a higher increase than those in wild-type mice [(119.594 ± 6.945) ng/L,(124.93-± 11.001 7) ng/L respectively,P ＜ 0.05].Moreover,plasma levels of corticosterone [(40.138 ±-0.805) μg/L] at 4 hours and [(37.440-0.485)μg/L] at 24 hours in gene knock-out mice showed a same result as compared with that in wild-type mice [(19.702 ± 0.804) μg/L,(17.602 ± 0.743) μg/L respectively,P ＜ 0.05].Conclusions Knock-out of adenosine A2A receptor increases the release of ACTH and corticosterone in acute stage of moderate craniocerebral trauma and promotes pituitary-adrenal stress response.This may provide a novel explanation for the neuroprotective effect of A2A receptor deficiency.

Objective To investigate the effect of the multimodal analgesia on postoperative pain after renal transplantation and the cytokines .Methods 40 cases of allogaft renal transplantation due to chronic renal failure were randomly divided into two groups (n=20) .The group D received the multimodal analgesia :preemptive analgesia plus patient controlled epidural analgesia(PCEA) and the group C(control) received analgesic drugs by intermittent intramuscular injection .The visual analogue scale(VAS) scores , the Ramsay sedation scores ,HR ,MAP and SPO2 at postoperative 2 ,6 ,12 ,24 ,48 h were recorded .Blood interleukin-2(IL-2) ,in-terleukin-6(IL-6) and interleukin-10(IL-10) levels were measured before anesthesia ,at the end of operation and postoperative 6 , 24 ,48 h .Results Postoperative MAP and SPO2 had no obvious change in the two groups ,no statistical differences in the various time points existed between the two groups (P>0 .05) .HR was significantly increased at 6 ,24 h after operation in the group C , which had statistical difference compared with that at the same time points in the group D (P<0 .05) .The VAS scores at postoper-ative 6 ,12 ,24 h in the group D were significantly lower than those in the group C ,the difference showed statistical significance (P<0 .05) .The sedation scores at various time points had no statistical difference between the two groups (P>0 .05) .The levels of IL-2 and IL-10 at postoperative 6 ,24 ,48 h in the two groups were significantly higher than those before anesthesia and at the end of operation (P<0 .05) .The levels of IL-2 and IL-6 at postoperative 6 ,24 ,48 h in the group D were significantly lower than those in the group C(P<0 .05) .Conclusion Multimodal analgesia can reach the effective analgesic effect ,down-regulate the pro-inflam-matory cytokines and up-regulate anti-inflammatory cytokines for maintaining postaperative serum cytokines balance .