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Objective:To explore the effect of hyperbaric oxygen (HBO) on the blood-brain barrier via the silent information regulator 1 (SIRT1)/Forkhead box O1(FoxO1) signaling pathway after cerebral ischemia and reperfusion using a rat model.Methods:Forty Wistar rats were randomly assigned into sham, cerebral ischemia-reperfusion (CIR), CIR+ HBO and CIR+ HBO+ EX527 groups, each of 10. The cerebral ischemia-reperfusion model was established in all groups except the sham group by right middle cerebral artery occlusion using the modified thread-occlusion method. The sham group was not ligated. Both the CIR+ HBO and CIR+ HBO+ EX527 groups were given HBO 1, 9, 21, 45 and 69 hours after the reperfusion. The CIR+ HBO+ EX527 group was additionally injected with 5mg/kg of EX527(a SIRT1inhibitor) peritoneally 4, 12, 24, 48 and 72 hours after the reperfusion. Then 2% Evens blue (EB) was injected into the tail vein an hour before the rats were sacrificed. The content of EB and the expression of SIRT1, FoxO1, ZO-1, Occludin, Claudin-5 mRNA and their proteins were determined using spectrophotometry, reverse transcription-polymerase chain reactions and Western blotting.Results:The average EB content of the hippocampal brain tissue from the CIR, CIR+ HBO and CIR+ HBO+ EX527 rats was significantly greater than the Sham group′s average 72h after reperfusion. The average expression of SIRT1, FoxO1, ZO-1, Occludin and Claudin-5 mRNA and their proteins was significantly lower, with the CIR + HBO + EX 527 group′s average significantly lower than that of the CIR+ HBO group.Conclusions:HBO can increase the expression of tight junction protein via the SIRT1/FoxO1 pathway. It helps to protect the blood-brain barrier in CIR injury situations.
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Objective To evaluate the role of glycogen synthase kinase 3 beta (GSK-3β) in lipid emulsion-induced inhibition of bupivacaine-induced apoptosis in cardiomyocytes of rats using RNA interference (RNAi) adenovirus infection method.Methods H9C2 cells were transferred into 96-well cell plates at a density of 1× 105 cells/ml after culture and then divided into 8 groups (n =10 each) using a random number table:control group (group C),bupivacaine group (group B),lipid emulsion group (group LE),bupivacaine plus lipid emulsion group (group B+LE),control plus GSK-3βRNAi adenovirus (GSK-3βi) group (group C+GSK-3βi),bupivacaine plus GSK-3βi group (group B+GSK-3βi),lipid emulsion plus GSK-3βi group (group LE+GSK-3βi) and bupivacaine plus lipid emulsion plus GSK-3βi group (group B+LE+GSK-3βi).ln B,LE and B+LE groups,the cells were incubated with culture medium containing 1 mmol/L bupivacaine,1％ lipid emulsion and 1 mmol/L bupivacaine plus 1％ lipid emulsion,respectively.In C+GSK-3βi,B+GSK-3βi,LE+GSK-3βi and B+LE+GSK-3βi groups,the cells were incubated with the drugs mentioned above on 2nd day after being infected by adenovirus.At 24 h after incubation with drugs,the expression of Bax and Bcl-2 was determined by Western blot,and the apoptosis rate was calculated using DAPI staining.Results Compared with group C,the expression of Bax was significantly upregulated,the expression of Bcl-2 was down-regulated,and the apoptosis rate was increased in group B (P＜0.05).Compared with group B,the expression of Bax was significantly down-regulated,the expression of Bcl-2 was up-regulated,and the apoptosis rate was decreased in group B+LE (P＜0.05).Compared with group B+LE,the expression of Bax was significantly up-regulated,the expression of Bcl-2 was downregulated and the apoptosis rate was increased in group B+LE+GSK-3βi (P＜0.05).Conclusion The mechanism by which lipid emulsion inhibits bupivacaine-induced apoptosis in cardiomyocytes of rats is associated with GSK-33.
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Objective To evaluate the effect of bupivacaine on miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) of neurons in hippocampal CA1 region of rats. Methods Twenty SPF healthy male Sprague-Dawley rats, aged 4-6 weeks, weighing 300-350 g, were divided into control group ( C group, n=10) and bupivacaine model group ( BPV group, n=10) using a random number table method. The central nervous system toxicity model was established by infusing 0. 75％ bupivacaine at a rate of 1 mg·kg-1 ·min-1 via the tail vein in BPV group, while normal saline was infused for 7. 5 min at a rate of 1 mg·kg-1 ·min-1 via the tail vein in C group. Rats were sacri-ficed at 6 h after successful establishment of the model, the brains was removed and hippocampal slices were prepared. The whole-cell patch-clamp technique was used to record the frequency and amplitude of mEPSCs and mIPSCs. Results Compared with C group, the frequency of mIPSCs was significantly de-creased ( P<0. 01) , and no significant change was found in the amplitude of mEPSCs or frequency and am-plitude of mIPSCs in BPV group ( P>0. 05) . Conclusion The mechanism of bupivacaine-induced central nervous system toxicity is related to decreasing the frequency of mIPSCs in hippocampal neurons of rats.
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Objective To investigate the effect of lipid emulsion on mitochondrial energy metabolism during bupivacaine-induced myocardiotoxicity in rats.Methods H9c2 cells were inoculated in 96-well plates at a density of 1 × 105cells/ml, and were randomly divided into 4 groups (n =18 each) using a random number table: control group (group C) , bupivacaine group (group B) , lipid emulsion group (group LE) , and bupivacaine + lipid emulsion group (group B + LE).The cells were incubated in the normal culture medium in group C.In group LE, the cells were incubated in the culture medium containing 1％ lipid emulsion.In group B, the cells were incubated in the culture medium containing 1 mmol/L bupivacaine.In group B + LE, the cells were incubated in the culture medium containing 1 mmol/L bupivacaine and 1％ lipid emulsion.After 24 h of incubation, the contents of ATP, ADP, and AMP were measured by high-performance liquid chromatography, and apoptotic rate was calculated by Hoechst33342/ PI staining.Results Compared with group C, the contents of ATP, ADP and AMP were significantly decreased, and apoptotic rate was increased in group B (P＜0.05), and the contents of ATP and ADP in group LE and ATP content in group B + LE were increased, and no significant changes were found in apoptotic rate in LE and B+LE groups (P＞0.05).Compared with group C, the contents of ATP, ADP and AMP were significantly increased, and apoptotic rate was decreased in LE and B+LE groups (P＜ 0.05).Compared with group LE, the contents of ATP, ADP and AMP were significantly decreased (P＜ 0.05), and no significant change was found in apoptotic rate in group B+LE (P＞0.05).Conclusion The mechanism by which lipid emulsion reduces bupivacaine-induced myocardiotoxicity may be associated with improved mitochondrial energy metabolism in rats.
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Objective To evaluate the effect of mitochondrial permeability transition pore (mPTP) in lipid emulsion-induced inversion of bupivacaine myocardiotoxicity in rats.Methods H9c2 cells were inoculated in 6-well plates at a density of 105 cells/ml, and were randomly divided into 4 groups (6 wells in each group, 2 ml/well) using a random number table: control group (group C) , bupivacaine group (group B) , lipid emusion + bupivacaine group (group LB) , and lipid emusion + bupivacaine + atractyloside group (group LBA).Phosphate buffer solution 100 μl was added to the culture medium in group C.In group B, bupivacaine was added to the culture medium with the final concentration of 1 mmol/L.In group LB, lipid emusion and bupivacaine were added to the culture medium with the final concentrations of 1％ and 1 mmol/L, respectively.In group LBA, lipid emusion, bupivacaine and atractyloside (an mPTP opener) were added to the culture medium with the final concentrations of 1％, 1 mmol/L and 30 μmol/L, respectively.All the cells were incubated for 24 h.After the end of incubation, the expression of Bcl-2, Bax, phosphorylated Bad (p-Bad) , caspase-3, activated caspase-3, caspase-9,activated caspase-9 and cytochrome c (Cyt c) was detected using Western blot.The expression of Bcl-2 mRNA, Bax mRNA, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was detected using real-time reverse transcriptase polymerase chain reaction.The ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/Bcl-2 mRNA were calculated.Results Compared with group C,the ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/ Bcl-2 mRNA were significantly increased, the expression of p-Bad was down-regulated, and the expression of Cyt c, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was up-regulated in group B (P＜0.05) , and no significant change was found in the parameters mentioned above in group LB (P＞0.05).Compared with group B, the ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/Bcl-2 mRNA were significantly decreased, the expression of p-Bad was up-regulated, and the expression of Cyt c, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was down-regulated in LB and LBA groups (P＜ 0.05).Compared with group LB, the ratios of Bax/Bcl-2, activated caspase-3/caspase-3, activated caspase-9/caspase-9, and Bax mRNA/Bcl-2 mRNA were significantly increased, the expression of p-Bad was down-regulated, and the expression of Cyt c, Bad mRNA, caspase-9 mRNA and Cyt c mRNA was up-regulated in group LBA (P ＜ 0.05).Conclusion The mechanism underlying lipid emulsioninduced inversion of bupivacaine myocardiotoxicity is related to inhibited mPTP opening in rats.
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Objective To investigate the effects of intrathecal (IT) lidocaine on propofol-induced sedation and content of lidocaine in brain tissues in rats.Methods Twenty male Sprague-Dawley rats,aged 2-3 months,weighing 250-350 g,were equally and randomly assigned to one of four groups:control group (group C),iv lidocaine group (group IV-L),IT normal saline group (group IT-NS) and IT lidocaine group (group IT-L).Groups IT-NS and IT-L received IT normal saline 15μl and 2％ lidocaine 15 μl,respectively.Group IV-L received iv 2％ lidocaine 15 μl.Propofol was infused starting from 10 min after IT or iv administration.When the eyelash reflex disappeared,the infusion of propofol was stopped and the dose of propofol consumed was recorded.The rats were sacrificed in IT-L and IV-L groups and brains were removed for determination of lidocaine level in brain tissues (by RP-HPLC).Results Compared with groups C,IV-L and IT-NS,the dose of propofol consumed when the eyelash reflex disappeared was significantly decreased in group IT-L (P ＜ 0.05).No significant difference was found in the propofol requirement when the eyelash reflex disappeared between groups C,IV-L and IT-NS and in the content of lidocaine in brain tissues between groups IV-L and IT-L (P ＞ 0.05).Conclusion Sedation induced by lidocaine administered intrathecally is not due to a direct action of lidocaine on the brain in rats.
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Objective To investigate the effects of hyperbaric oxygen (HBO) on the expression of caveolin2 and matrix-metalloproteinase-9 (MMP-9) in brain tissues and the blood brain barrier (BBB) after cerebral ischemia and reperfusion (I/R) in rats. Methods Three hundred and seventy male Wistar rats were randomly assigned into sham,ischemia-reperfusion,HBO,and I/R + HBO groups. After creating cerebral I/R models,oxygen at 0.25 MPa was administered 5 times,and 2％ Evans blue (EB) was injected into the tail veins 1 h before the rats were sacrificed.The permeability of the BBB,the expression of caveolin-2 and MMP-9,and EB content were determined by Western blotting,immunohistochemistry,and spectrophotometery,respectively. Results In the I/R group,the EB content increased steadily to a peak at 4 hours.EB content in the IR + HBO group was significantly lower than in the I/R group.Caveolin-2 and MMP-9 were significantly augmented by I/R injury at the 24th,48th and 72nd hours.Compared to the I/R group,HBO intervention decreased their expression levels. Conclusion HBO intervention can reverse the increase of caveolin-2 and MMP-9 caused by I/R injury,which suggests a mechanism for protective effects of HBO on the permeability of the BBB in I/R injury.
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Objective To investigate the effect of liver cirrhosis on the potency of propofol for sedation in rats. Methods Fifty-eight male SD rats, aged 10-12 weeks, weighing 180-220 g, were randomly divided into 3 groups: control group (group C, n = 18), mild liver cirrhosis group (group M1, n =20) and severe liver cirrhosis group (group M2, n = 20). The model of liver cirrhosis was established using four factors described by Chen et al. After successful establishment of the model, propofol was injected intravenously. The dose of propofol was determined by up-and-down sequential method for loss of righting reflex. The dose of propofol was 5.912 mg/kg in the first rat and the ratio of the doses between the two consecutive rats was 0.85. ED50 of propofol was calculated using up-and-down sequential method. Results ED50 of propofol was significantly lower in group M1 and M2 than in group C and in group M2 than in group M1 ( P ＜ 0.05 or 0.01 ). Conclusion The liver cirrhosis can enhance the potency of propofol for sedation in rats.
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Objective To investigate the effects of remifentanil on lipid peroxidation druing hemorrhagic shock-induced acute lung injury (ALI) in rabbits. Methods Thirty-two healthy adult rabbits weighing 2.0-2.5 kg were randomly divided into 4 groups (n = 8 each): sham operation group (group S); ALI group; low-dose remifentanil group (group LR); high-dose remifentanil group (group HR). The left femoral artery was cannulated for blood-letting and blood sampling. The right femoral artery was cannulated for remifentanil administration. The model of hemorrhagic shock was established by modified Wigger' s methods. In group S, only cannulation was performed. In group LR and HR, remifentanil was infused intraperitoneally at 0.66 and 1.32 μg·kg-1 ·min-1 15 min before blood-letting respectively, while group ALI received equal volume of normal saline instead. Arterial blood samples were taken at 0, 20,70 and 100 min after blood-letting (T1-4) for blood gas analysis. The animals were then sacrificed and the lungs were immediately removed for histological examination with light microscope and determination of W/D lung weight ratio, MDA content and SOD activity. Results W/D ratio and MDA content were significantly increased, while SOD activity was significantly decreased in group ALI compared with group S (P ＜0.05). The pH value at T2 and PaO2 at T2-4 in group LR and the pH value and PaCO2 at T2-4 in group HR were significantly higher than those in group ALI (P ＜ 0.05). W/D ratio and MDA content were significantly lower,while SOD activity was significantly higher in group LR and HR than in group ALI, and in group HR than in group LR (P ＜ 0.05). Remifentanil infusion significantly attenuated the pathologic changes in a dose-dependent manner. Conclusion Remifentanil pretreatment can attenuate hemorrhagic shock-induced ALI through inhibiting lipid peroxidation in rabbits.
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Objective To investigate the effects of hyperbaric oxygen(HBO)on the expression of MMP-9 and MMP-2 mRNA in the brain tissues after cerebral ischemia and reperfusion,and the permeability of blood brain barrier(BBB).Methods Using cerebral ischemia-reperfusion models with conscious mice,0.25 MPa(atmosphaera absolutus,ATA)HBO was applied 5 times during the reperfusion period,and 2%Evan's blue(EB)was injected into the tail vein 1 hour before the animals were sacrificed.The expression of MMP-9 and MMP-2 mRNA and EB content were determined by RT-PCR and spectrophotometry.Results The expression of MMP-9 and MMP-2 mRNA aswell as EB content significantly increased in the ischemia-reperfusion group as compared with a sham surgery group.The expression of MMP-9 and MMP-2 mRNA,and EB levels in the HBO group were similar to those in the sham surgery group.The expression of MMP-9 and MMP-2 mRNA and EB levels in the group given HBO plus reperfusion group were significantly decreased as compared with those in the group receiving reperfusion alone. Conclusion HBO can significandy reduce the expression of MMP-9 and MMP-2 mRNA and the permeability of the BBB.
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Objective To explore the effect of hyperbaric oxygenation (HBO) on generation of free radicals and the permeability of blood-brain barrier(BBB) during cerebral ischemia-reperfusion(CIR). Methods Three hundred and twenty Kunming mice were randomly divided into four groups: a sham operation group, a HBO group, a CIR group, and a HBO + CIR group, with 80 mice in each group. Conscious mice were used to establish the CIR model, and 0.25MPa (ATA) HBO were applied 5 times after operations. The activities of SOD, CAT, GSH-PX and the concentration of MDA, EB in the cerebral hippocampal tissues in each group were measured with colorimetry. The cerebral hippocampal tissues were harvested and processed, then observed and compared with transmission electron microscopic observation. Results Compared with the sham operation group, the activities of antioxidation enzymes (GSH-PX,SOD, CAT) in CIR group decreased significantly (P
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AIM: To study effects of hyperbaric oxygen (HBO) and cyclosporin A (CsA) on the contents of active oxygens and nitric oxide (NO) in spleens of skin transplanted mice.METHODS: The donor mice BALB/C and receptor mice C 57BL/6 were tested for skin transplantation. The HBO group mice were treated with 99.2% oxygen under 0.25 MPa for 1.5 hours, while CsA group mice were treated with CsA 0.5 mg?kg -1?d by abdomen injection. After 14 days, the spleen were extracted the contents of malondialdehyde (MDA) and NO and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), catalase (CAT) and NO synthases (NOS) were determined.RESULTS: (1) Compared with the control group, the transplantation group, HBO group and CsA group have markedly increased the content of MDA and the activities of GSH-PX and CAT; Compared with the transplantation group, the CsA group have markedly increased activity of SOD and reduced activities of GSH-PX and CAT; the HBO group have markedly reduced the activity of GSH-PX and increased the activities of CAT and SOD (P
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AIM: To study effects of hyperbaric oxygen (HBO) and cyclosporin A (CsA) on the contents of active oxygens and nitric oxide (NO) in spleens of skin transplanted mice. METHODS: The donor mice BALB/C and receptor mice Cs7BL/6 were tested for skin transplantation. The HBO group mice were,treated with 99.2 % oxygen under 0.25 MPa for 1.5 hours, while CsA group mice were treated with CsA 0.5 rmg· kg- t· d- 1 by abdomen injection. After 14 days, the spleen were extracted the contents of malondialdehyde (MDA) and NO and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH - PX), catalase (CAT) and NO synthases (NOS) were determined. RESULTS: (1) Compared with the control group, the transplantation group, HBO group and CsA group have markedly increased the content of MDA and the activities of GSH - PX and CAT; Compared with the transplantation group, the CsA group have markedly increased activity of SOD and reduced activities of GSH - PX and CAT; the HBO group have markedly reduced the activity of GSH - PX and increased the activities of CAT and SOD (P < 0.01 ). (2) Compared with the control group, the transplantation group have markedly increased the content of NO and the activity of NOS; Compared with the transplantation group, the HBO group have markedly increased the activity of NOS and reduced the content of NO ( P < 0.01 ); The content of NO and the activity of NOS in CsA group was not changed significantly. CONCLUSION: In the lymphocytes of the transplantation group, the peroxidation is intensified, and the content of NO and the activity of NOS increased. HBO and CsA may activate the systems of oxidation/antioxidation and NO/NOS in spleen, which may be related to their mechanism of inhibition rejection.
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Objective To estimate the relationship between the parameters used to estimate the depth of anesthesiaMethods Fifty-two ASA I - II patients undergoing choleeystectomy or exploration of eommon bile duet without jaundice were emdled in the study. Premedieation consisted of midazolam 5 mg and atropine 0.5 mg im.30 min before operation. Anesthesia was induced with fentanyl 4 ug.kg-1 , droperidol 0.08 mg.kg-1 , propofol 2 mg. kg-1 and vecuronium 0.1 mg.kg-1 , and maintained with enflurane and continuous infusion of propofol and intermittent intravenous boluses of vecuronium. The patients were intubated and mechanically ventilated. B1S,HRV and BP were continuously monitored and recorded before induction (T1 ) , 1 min(T2 ) , 3 min(T3 ) after intubation, 1 min before skin incision (T4) , 3 min after skin incision (T5), 1 h after induction (T6), 1 min before extubation (T7) and when the patient was conscious (T8). Blood samples were taken at the same intervals for detenninaton of blood propofol and cortisol level (n = 18) by using radioimmunoasscey and HPL, BIS was maintained at 30 ~ 60 during anesthesia by adjustment of propofol infusion rate. Results There was negative correlation between plasma propofol concentration and BIS/MAP; there was positive correlation between HR and MAP. Plasma cortisol level was positively correlated with BIS, MAP and HR and negatively correlated with plasma propofol concentration. Conclusion The LF and HF can reflect the changes in cardiac sympathetic-vagal tension but cannot reflect the depth of anesthesia. Stress response can be controlled by plasma propofol concentration and estimated by BIS,MAP and HR monitoring.
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Objective To observe the effects of arsenic trioxide(As2O3)on the expressions of TNF-?,Fas and bcl-2 in lung adenocarcinoma cells(LAC)and to explore the mechanism of arsenic trioxide inducing apoptosis.Methods The expressions of TNF-?,Fas and bcl-2 in lung adenocarcinoma cells pretreated by arsenious acid were determined by the double antibody sandwich ABC-ELISA method.Results Compared with the control group,As2O3 showed no effects on the contents of bcl-2 in lung adenocarcinoma cells after 72 hours treatment,but increased the contents of TNF-? and Fas significantly,and the effects in different concentration groups had significant differences.The protein expressions of TNF-? and Fas showed a tendency of concentration-dependent increasing.Conclusions The results suggest that As2O3 induces the apoptosis of LAC cells possibly by up-regulating the expression of TNF-? and Fas.
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Objective To explore the molecular mechanism of Qingjin Desheng Tablets on inhibiting the growth of lung cancer cells and to supply experimental evidences for Chinese herbal medicine preventing and curing lung cancer in clinical application .Methods Immunohistochemistry staining was used to detect the activity of telomerase and the expression of bcl-2 and bax in human lung adenocarcinoma cells after treated with serum containing Qingjin Desheng Tablets.Results The activity of telomerase was inhibited significantly and the positive rate of bcl-2 protein reduced,and the positive rate of bax2 increased in human lung adenocarcinoma cells after 72 hour-treatment with the serum containing Qingjin Desheng Tablets.Conlusion Qingjin Desheng Tablets exert the anti-tumor action through inhibiting the activity of telomerace and modulating the expression of bcl-2 and bax protein.
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AIM: To investigate the effect of hyperbaric oxygen (HBO) on gelatinase,nitric oxide synthase and the permeability of brain blood barrier(BBB) in ischemia-reperfusion(I/R) mice. METHODS: Using cerebral I/R models, during the reperfusion period, 0.25 MPa (ATA) HBO were applied 5 times. matrix metalloproteinase(MMP)-2,9, nitric oxide synthase(NOS) and evans blue (EB) in brain were measured. RESULTS: ①HBO had significanty effect on MMP-9, but had little effect on MMP-2. ② HBO decreased the activity of NOS.③ The maxium amount of EB in IR group was at 4 hours after reperfusion and gradually decreased at 11 h, 23 h,48 h, 72 h. CONCLUSION: HBO may decrease the activities of MMP-9,NOS and the permeability of BBB in cerebral ischemia-reperfusion mice.