ABSTRACT
Objective:To evaluate the role of DNA methyltransferase in acute lung injury in septic mice.Methods:Forty-eight healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (group Sham), sham operation+ DNA methyltransferase inhibitor group (group Sham+ 5-Aza), sepsis group (group Sepsis) and sepsis+ DNA methyltransferase inhibitor group (group Sepsis+ 5-Aza). Sepsis model was developed by cecal ligation and puncture (CLP) in anesthetized mice.Mice were sacrificed at 24 h after CLP, and lung tissues were obtained, DNA was extracted to determine the global DNA methylation by colorimetry, and RNA was extracted to detect the expression of DNA methyltransferase (DNMTl, DNMT3a, DNMT3b) mRNA by real-time fluorescent quantitative polymerase chain reaction, the wet/dry lung weight ratio (W/D ratio) was measured, the histopathological changes of lung tissues were determined by HE staining, the contents of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), high-mobility group box 1 protein (HMGB1) and malondialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase were measured by enzyme-linked immunosorbent assay. Results:Compared with group Sham, the global DNA methylation was significantly increased, the expression of DNMT1 and DNMT3a mRNA was up-regulated, the lung injury score, W/D ratio, and contents of IL-6, TNF-α, HMGB1 and MDA were increased, and activities of SOD and CAT were decreased at 24 h after CLP in group Sepsis and group Sepsis+ 5-Aza ( P<0.05), and no significant change was found in the indexes mentioned above in group Sham+ 5-Aza ( P>0.05). Compared with group Sepsis, the global DNA methylation was significantly decreased, the expression of DNMT1 and DNMT3a mRNA was down-regulated, the lung injury score, W/D ratio, contents of IL-6, TNF-α, HMGB1 and MDA were decreased, and the activities of SOD and CAT were increased in group Sepsis+ 5-Aza ( P<0.05). Conclusions:DNA hypermethylation mediated by DNMT1 and DNMT3a is involved in the process of acute lung injury in septic mice.
ABSTRACT
Objective:To evaluate the effect of hydrogen-rich saline on serine threonine protein kinase (Akt) /nuclear factor E2-related factor 2 (Nrf2) signaling pathway during hypoxia/reoxygenation (H/R) injury to human renal tubular epithelial cells.Methods:Human renal tubular epithelial cell line were seeded in 96-well plates at a density of 1.5×10 4 cells/ml (200 μl/well) or in 6-well plates at a density of 2×10 5 cells/ml (2 ml/well) were divided into 5 groups( n=30 each) using a random number table method: control group (group C), hydrogen-rich group (group H), group H/R, H/R plus hydrogen-rich saline group (group H/R+ H) and H/R plus hydrogen-rich saline plus Akt inhibitor uprosertib group (group H/R+ H+ U) .In group C, the cells were incubated for 28 h in an incubator filled with normoxia at 37 ℃ (5%CO 2-21%O 2-74%N 2). In group H, cells were added to the medium containing 0.6 mmol/L hydrogen-rich saline, and then incubated for 28 h in an incubator filled with normoxia at 37 ℃.In group H/R, the cells were incubated in an anaerobic chamber (37 ℃, 5%CO 2-1%O 2-94 %N 2) for 24 h, and then incubated for 4 h in an incubator filled with normoxia at 37 ℃.In group H/R+ H, the cells were incubated in an anaerobic chamber for 24 h, and then incubated for 4 h in an incubator containing 0.6 mmol/L filled with normoxia at 37 ℃.In group H/R+ H+ U, the cells were incubated for 1 h in the culture medium containing uprosertib 10 μmol/L (final concentration) and the other treatments were similar to those previously described in group H/R+ H. After treatment in each group, the cell viability was measured by MTT assay, cell apoptosis was measured using flow cytometry, superoxide dismutase (SOD) activity was measured using xanthine oxidase method), malondialdehyde (MDA) content was detected by thiobarbituric acid method, the expression of Akt, phosphorylated Akt (p-Akt), total Nrf2, nuclear Nrf2 and activated caspase-3 was detected by Western blot, and the expression of Nrf2 mRNA was detected by Real-time PCR. Results:Compared with group C, the cell viability and activity of SOD were significantly decreased, the apoptosis rate and content of MDA were increased, and the expression of p-Akt, nuclear Nrf2, total Nrf2, activated caspase-3 protein and Nrf2 mRNA was up-regulated in group H/R and group H/R+ H ( P<0.05). Compared with group H/R, the cell viability and activity of SOD were significantly increased, the apoptosis rate and content of MDA were decreased, the expression of p-Akt, nuclear Nrf2, total Nrf2 and Nrf2 mRNA was up-regulated and expression of activated caspase-3 protein was down-regulated in group H/R+ H ( P<0.05). Compared with group H/R+ H, the cell viability and activity of SOD was significantly decreased, the apoptosis rate and content of MDA were increased, the expression of p-Akt, nuclear Nrf2, total Nrf2 protein and Nrf2m RNA was down-regulated, and the expression of activated caspase-3 protein was up-regulated in group H/R+ H+ U ( P<0.05). Conclusion:The mechanism by which hydrogen-rich saline attenuates H/R injury to human renal tubular epithelial cells is related to improving activation of Akt/Nrf2 signaling pathway, decreasing oxidative stress response and inhibiting cell apoptosis.
ABSTRACT
Objective:To evaluate the role of nuclear factor erythroid 2-related factor/ heme oxygenase-1 (Nrf2/HO-1) signaling pathway in dexmedetomidine-induced reduction of oxygen-glucose deprivation and restoration (OGD/R) injury to microglia.Methods:BV-2 microglia were cultured in high-glucose DMEM culture medium supplemented with 10% fetal bovine serum in an normal culture incubator at 37 ℃ (5%CO 2-21%O 2-74 %N 2). The cells were seeded in 96-well plates at a density of 1.5×10 4 cells/ml (200 μl/well) or 6-well plates at a density of 2×10 5 cells/ml (2 ml/well) and divided into 5 groups ( n=30 each) using a random number table method: control group (group C), dexmedetomidine group (group D), group OGD/R, OGD/R+ dexmedetomidine group (group OGD/R+ D) and OGD/R+ dexmedetomidine+ ML385 group (group OGD/R+ D+ ML). The cells in group C were continuously cultured in a normal culture incubator for 26 h. In group D, dexmedetomidine at the final concentration of 10 μmol/L was added, cells were incubated for 2 h, and then were continuously incubated in a normal culture incubator for 26 h. In OGD/R, OGD/R+ D and OGD/R+ D+ ML groups, the culture medium was replaced with glucose-free DMEM culture medium, cells were cultured for 2 h in an incubator at 37 ℃ (5%CO 2-1%O 2-94 %N 2), the culture medium was replaced with high-glucose DMEM culture medium containing 10% fetal bovine serum and then the cells were cultured for 24 h in a normal incubator.Dexmedetomidine at the final concentration of 10 μmol/L was added at 2 h before OGD in OGD/R+ D and OGD/R+ D+ ML groups.Nrf-2 inhibitor ML385 at the final concentration of 4 μmol/L was added at 30 min before dexmedetomidine was added in group OGD/R+ D+ ML.Cells in 6 wells in each group were selected randomly for assessment of cell viability (by methyl thiazolyl tetrazolium assay) and apoptosis (using flow cytometry), and for determination of the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 in the supernatant (using enzyme-linked immunosorbent assay), the expression of Nrf2 in nucleus, Nrf2 and HO-1(by Western blot ) and the expression of HO-1 mRNA (by real-time polymerase chain reaction). Results:Compared with group C, the cell viability was significantly decreased, cell apoptosis rate and concentrations of TNF-α, IL-6 and IL-10 in the supernatant were increased, and the expression of Nrf2 in nucleus, Nrf2, HO-1 and its mRNA was up-regulated in OGD/R and OGD/R+ D groups ( P<0.05), and no significant change was found in each parameter mentioned above in group D ( P>0.05). Compared with group OGD/R, the cell viability and IL-10 in the supernatant concentration were significantly increased, cell apoptosis rate and concentrations of TNF-α and IL-6 in the supernatant were decreased and the expression of Nrf2 in nucleus, Nrf2, HO-1 and its mRNA was up-regulated in group OGD/R+ D ( P<0.05), and no significant changes were found in the parameters mentioned above in group OGD/R+ D+ ML ( P>0.05). Compared with group OGD/R+ D, the cell viability and concentration of IL-10 in the supernatant were significantly decreased, cell apoptosis rate and concentrations of TNF-α and IL-6 in the supernatant were increased and the expression of Nrf2 in nucleus, Nrf2, HO-1 and its mRNA was down-regulated in group OGD/R+ D+ ML ( P<0.05). Conclusion:The mechanism by which dexmedetomidine alleviates OGD/R injury to microglia may be related to promoting the activation of Nrf2/HO-1 signaling pathway and inhibition of inflammatory responses.
ABSTRACT
Peripheral nerve injury (PNI) is an important clinical complication, which brings long-term physical and psychological pain and economic burden to patients. There is no satisfactory treatment plan for PNI. Although microsurgery technology has been greatly developed, some peripheral nerve defects or ruptures caused by external forces can be repaired by surgery or nerve transplantation. However, due to the weak ability of nerve cell regeneration and surgical operations may cause damage to the injured nerves, the patient's functional recovery may not be able to achieve the desired effect. Therefore, it is urgent to find a safe and effective method to treat PNI. Mesenchymal stem cells have special differentiation potential and can differentiate into a variety of cell types in vitro and in vivo, and have received widespread attention from researchers. In this paper, the research progress of mesenchymal stem cells in nerve injury repair was summarized, and the characteristics, functions of mesenchymal stem cells and the mechanism of action in peripheral nerve injury repair were reviewed.
ABSTRACT
Objective To evaluate the role of DNA methylation in sepsis-associated encephalopathy in mice.Methods A total of 144 clean-grade healthy male C57BL/6 mice,aged 6-8 weeks,weighing 20 -25 g,were divided into 4 groups (n=36 each) using a random number table method:sham operation group (group Sham),sepsis group (group Sepsis),sham operation plus S-adenosyl methionine (SAM) group (group Sham+SAM) and sepsis plus SAM group (group Sepsis+SAM).Sepsis was produced by cecum ligation and puncture (CLP).In Sham+SAM and Sepsis+SAM groups,DNA methylated methyl donor SAM 100 mg/kg was intraperitoneally injected at 1 h before operation and 12 h after operation,while the equal volume of normal saline was given instead in Sham and Sepsis groups.The cognitive function was assessed using Y-maze and contextual fear conditioning test at 1,3 and 7 days after CLP.Mice were sacrificed at 1,3 and 7 days after CLP,and the hippocampal tissues were taken for determination of genomewide DNA methylation (by colorimetric assay) and expression of DNA methyltransferase enzymes (DNMT1,DNMT3a,and DNMT3b),ten-eleven translocation (TET) enzymes (TET1,TET2 and TET3) and thymine-DNA glycosylase (TDG) mRNA (by fluorescent quantitative real-time).Results Compared with group Sham,the time of staying at the novel arm was significantly shortened,the percentage of time spent freezing and the total number of entries into each arm were reduced,genome-wide DNA methylation in hippocampal tissues was decreased at 1,3 and 7 days after CLP,the expression of DNMT1,DNMT3a,TET1,TET2,TET3 and TDG was up-regulated,and the expression of DNMT3b was down-regulated in group Sepsis (P＜0.05).Compared with group Sepsis,the time of staying at the novel arm was significantly prolonged,the percentage of time spent freezing and the total number of entries into each arm were increased,the expression of DNMT1,DNMT3a,TET1,TET2,TET3 and TDG mRNA was down-regulated,and the expression of DNMT3b was up-regulated in group Sepsis+SAM (P＜0.05).Conclusion DNA methylation is involved in the development of sepsis-associated encephalopathy in mice.
ABSTRACT
Objective@#To evaluate the role of DNA methylation in sepsis-associated encephalopathy in mice.@*Methods@#A total of 144 clean-grade healthy male C57BL/6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups (n=36 each) using a random number table method: sham operation group (group Sham), sepsis group (group Sepsis), sham operation plus S-adenosyl methionine (SAM) group (group Sham+ SAM) and sepsis plus SAM group (group Sepsis+ SAM). Sepsis was produced by cecum ligation and puncture (CLP). In Sham+ SAM and Sepsis+ SAM groups, DNA methylated methyl donor SAM 100 mg/kg was intraperitoneally injected at 1 h before operation and 12 h after operation, while the equal volume of normal saline was given instead in Sham and Sepsis groups.The cognitive function was assessed using Y-maze and contextual fear conditioning test at 1, 3 and 7 days after CLP.Mice were sacrificed at 1, 3 and 7 days after CLP, and the hippocampal tissues were taken for determination of genome-wide DNA methylation (by colorimetric assay) and expression of DNA methyltransferase enzymes (DNMT1, DNMT3a, and DNMT3b), ten-eleven translocation (TET) enzymes (TET1, TET2 and TET3) and thymine-DNA glycosylase (TDG) mRNA (by fluorescent quantitative real-time).@*Results@#Compared with group Sham, the time of staying at the novel arm was significantly shortened, the percentage of time spent freezing and the total number of entries into each arm were reduced, genome-wide DNA methylation in hippocampal tissues was decreased at 1, 3 and 7 days after CLP, the expression of DNMT1, DNMT3a, TET1, TET2, TET3 and TDG was up-regulated, and the expression of DNMT3b was down-regulated in group Sepsis (P<0.05). Compared with group Sepsis, the time of staying at the novel arm was significantly prolonged, the percentage of time spent freezing and the total number of entries into each arm were increased, the expression of DNMT1, DNMT3a, TET1, TET2, TET3 and TDG mRNA was down-regulated, and the expression of DNMT3b was up-regulated in group Sepsis+ SAM(P<0.05).@*Conclusion@#DNA methylation is involved in the development of sepsis-associated encephalopathy in mice.
ABSTRACT
Objective To investigate the effect of dexmedetomidine on the expression of hypoxia-inducible factor-1α (HIF-1α) during hypoxia/reoxygenation (H/R) in human renal tubular epithelial cells.Methods Human renal tubular epithelial cells (HK-2 cells) cultured in vitro were randomly divided into 4 groups (n =24 each) using a random number table:control group (group C),dexmedetomidine group (group DEX),H/R group and H/R+ dexmedetomidine group (group H/R + DEX).In group C,the cells were incubated for 28 h in an incubator filled with normoxia at 37 ℃.In group DEX,dexmedetomidine 0.1 nmol/L (final concentration) was added to the culture medium and the cells were incubated for 2 h,and then incubated for 28 h in an incubator filled with normoxia at 37 ℃.In group H/R,the cells were incubated in an anaerobic chamber for 24 h at 37 ℃,and then incubated for 4 h in an incubator filled with normoxia at 37 ℃.In group H/R + DEX,the cells were incubated for 2 h in the culture medium containing dexmedetomidine 0.1 nmol/L (final concentration),incubated in an anaerobic chamber for 24 h at 37 ℃,and then incubated for 4 h in an incubator filled with normoxia at 37 ℃.After treatment in each group,the cell viability was measured by MTT assay,cell apoptosis was measured using flow cytometry,the expression of HIF-1α mRNA was detected using RT-PCR,the expression of HIF-1α and activated caspase-3 protein was detected by Western blot,and the cell growth was observed.The apoptosis rate was calculated.Results Compared with group C,the cell viability was significantly decreased,the apoptosis rate was increased,and the expression of HIF-1α mRNA and protein and activated caspase-3 protein was up-regulated in H/.R and H/R + DEX groups,and no significant change was found in group DEX.Compared with group H/R,the cell viability was significantly increased,the apoptosis rate was decreased,the expression of HIF-1α mRNA and protein was up-regulated,the expression of activated caspase-3 protein was down-regulated,and the cell status was significantly improved in group H/R + DEX.Conclusion The mechanism by which dexmedetomidine attenuates H/ R-induced damage to human renal tubular epithelial cells may be related to up-regulated expression of HIF-1 α and inhibited cell apoptosis.
ABSTRACT
Objective To investigate the effects of different doses of dexmedetomidine on the minimum alveolar concentration (MAC) of sevoflurane for sedation in patients.Methods ASA physical status Ⅰ or Ⅱ patients of both sexes,aged 18-64 yr,undergoing elective lower abdominal surgery performed under general anesthesia,were randomly divided into 4 groups:control group (group C) and different doses of dexmedetomidine groups (D1,D2 and D3 groups).In D1,D2 and D3 groups,the loading dose of dexmedetomidine 0.4,0.6 and 0.8 μg/ kg was intravenously infused over 15 min,respectively,adverse cardiovascular events were then recorded,followed by infusion at 0.4,0.6 and 0.8 μg· kg-1 · h-1 via a pump,respectively,while in group C,the equal volume of 0.9 ％ normal saline was given instead of dexmedetomidine.Sevoflurane administration was begun after completion of infusion of the loading dose.Up-and-down sequential allocation was used to determine the MAC.The initial end-tidal concentration of sevoflurane was set at 0.8％,0.7％,0.6％ and 0.5％ in C,D1,D2 and D3 groups,respectively,and maintained at this level for 15 min.Each time the concentration of sevoflurane increased/decreased in the next patient depending on whether or not the patients correctly followed the verbal command to open his eyes.The ratio between the two consecutive concentrations was 0.9.The middle point between the positive response and negative response served as a crossover pair.After at least 7 independent crossover pairs were observed in each group,the experiment was stopped.The MAC and 95 ％ confidence interval of sevoflurane were calculated.Results The incidence of adverse cardiovascular events was significantly higher in D3 group than in D1 and D2 groups (P ＜ 0.05).In C,D1,D2 and D3 groups,the MAC (95％ confidence interval) of sevoflurane was 0.68％ (0.64％-0.74％),0.50％ (0.47％-0.52％),0.36％ (0.32％-0.41％) and 0.28％(0.26％-0.31％),respectively.The MAC-awake of sevoflurane was significantly lower in D1-3 groups than in group C,in D2 and D3 groups than in group D1,and in D3 group than in group D2 (P ＜ 0.05).Conclusion Dexmedetomidine 0.6μg/kg can significantly decrease the MAC of sevoflurane for sedation,induces no side effects and is the optimum dose in patients.
ABSTRACT
Objective To evaluate the effects of different doses of dexmedetomidine on the median effective concentration (EC50) of propofol given by target-controlled infusion (TCI) at loss of consciousness (LOC).Methods Eighty ASA Ⅰ or Ⅱ patients of both sexes,aged 18-64 yr,with body mass index ≤25 kg/m2,scheduled for operations under general anesthesia,were randomly allocated to one of four groups(n=20 each): control group (group C) and dexmedetomidine 0.4 μg/kg group (group D1),dexmedetomidine 0.5 μg/kg group (group D2) and dexmedetomidine 0.6 μg/kg group (group D3).Dexmedetomidine 0.4,0.5 and 0.6 μg/kg were infused intravenously over 10 min in groups D1-3,while the equal volume of normal saline was given instead of dexmedetomidine in group C.Propofol was then given by TCI and the EC50 was determined by up-and-down sequential trial.The target plasma concentration was set at 2.0μg/ml in the first patient in each group.The ratio of the target plasma concentration between the two consecutive patients was 1.1.Loss of response to eyelash stimulation and verbal command (2 times) was considered to be signs of LOC.The EC50 and 95％ confidence interval (CI) of propofol causing LOC were calculated.Complications such as bradycardia,hypotension and respiratory depression were recorded.Results The EC50 (95％ CI) of propofol causing LOC was 2.59 (2.51-2.67),2.09 (2.02-2.16),1.82 (1.70-1.95) and 1.60 (1.49-1.72) μg/ml in groups C and D1.3 respectively.The EC50 of propofol causing LOC was significantly lower in groups D1-3 than in group C.Dexmedetomidine significantly decreased the EC50 of propofol required for causing LOC in a dose-dependent manner in groups D1-3 (P ＜ 0.05).The incidences of bradycardia and hypotension were significantly lower in groups D1.3 than in group C (P ＜ 0.05).Compared with group D1,the incidence of bradycardia was increased in groups D2,3 and the incidence of hypotension was increased in group D3 (P ＜ 0.05),There was no significant difference in the incidences of bradycardia and hypotension between groups D2 and D3 (P ＞ 0.05).No patients developed respiratory depression.Conclusion The optimum dose for dexmedetomidine infused intravenously when combined with propofol given by TCI is 0.4 μg/kg and it can decrease the EC50 of propofol administered by TCI at LOC with no adverse reactions.
ABSTRACT
Objective To investigate the effect of STH-2 cardioplegic solution containing levosimendan on ischemia-reperfusion (I/R) injury in isolated rat hearts. Methods Thirty-two male Wistar rats weighing 250-300 gwere anesthetized with intraperitoneal 3% pentobarbital 30 mg/kg. The hearts were rapidly excised and perfused with oxygenated (95% O2-5% CO2) K-H solution for 30 min in a Langendorff apparatus and then divided into 4groups (n = 8 each) according to the composition of cardioplegic solution: group Ⅰ control (group C) was perfused with STH-2 cardioplegic solution; group Ⅱ , Ⅲ and Ⅳ were peffused with STH-2 cardioplegic solution containing levosimendan 0.03 μmol/L (L1), 0.3 μmol/L (L2) and levosimendan 0.3 μmol/L + glibenclamide 10 μmol/L (L2+ G) respectively. The isolated hearts were first perfused with different cardioplegic solutions for 2 h and then with K-H solution for 30 min. The coronary effluent was collected before ischemia (baseline) and at 10, 20 and 30 min of reperfusion for measurement of creatine kinase (CK) and lactate dehydrogenase (LDH)activities. Myocardial specimens were obtained from apex at 30 min of reperfusion for determination of myocardial ATP and MDA contents and SOD activity. Results Perfusion with STH-2 cardioplegic solution significantly increased CK and LDH activities and MDA content, and significantly decreased SOD activity. Levosinendan 0.03or 0.3 μmol/L significantly attenuated the cardioplegia-induced increase in LDH,CK and SOD activities and MDA content. The protective effects of levosimendan on myocardium against I/R injury were reversed by glibenclamide to some extent. Conclusion Levosimendan can protect myocardium from I/R injury in a dose-dependent manner by opening KATP channel.
ABSTRACT
[Objective]To study the difference in expression of RFC, GST-?, DHFRmRNA between human osteosarcoma U2-OS cell line and the MTX-resisitant variants U2-OS/R1-R3, and to investigate the significance in MTX resistance for human osteosarcoma. [Methods]Three resistant MTX human osteosarcoma cell lines were established by pulse exposure parental cell line(U2-OS) in gradually increased dose of MTX . The expression of RFC,GST-?,DHFRmRNA were assayed by real-time fluorescence quantitative polymerase chain reaction(FQ-PCR).[Results]Three MTX-resistant variants(U2-OS/R1-R3) were successfully established , the results of the FQ-PCR revealed that the MTX resistance was associated with the decreased expression of the RFC mRNA and increased expression of DHFR mRNA and GST-? mRNA.[Conclusion]The author investigated the MTX resistant mechanism of human osteosarcoma cell line at a gene level. The decreased expression of RFC mRNA and the increased expression of DHFR mRNA and GST-? mRNA participate in the MTX resistance in human osteosarcoma cell lines U-2 OS. This provides the evidence for exploring the MTX resistance mechanism in clinical osteosarcoma patients ,and helps to screen the patients who are insensitive to MTX chemotherapy.