Objective To investigate the effect of sevoflurane preconditioning combined with postconditioning (Spost) on anoxia/reoxygenation (A/R) injury to neonatal rat cardiomyocytes.Methods Primary cultured neonatal rat cardiomyocytes were isolated from SD rats aged 1-3 days and cultured in DMEM liquid culture medium.The cells were seeded in 24-well plates (1 ml/hole),35 mm diameter dishes (5 ml/dish) or in 50 mm culture flasks (8 ml/flask) with a density of 3 × 105/ml and randomly divided into 9 groups (n =24 each):control group (group C),A/R group,Spre group (group S1),Spre + SB203580 group (group S1 + SB),sevoflurane postcon-ditioning (Spost) group (group S2),Spost + SB203580 group(group S2 + SB),Spre + Spost group (group S3),Spre + Spost + SB203580 group (group S3 + SB),and group SB203580 (group SB).The cells were cultured routinely for 160 min in group C and the cells were exposed to 95％ N2-5％ CO2 in an incubator at 37 ℃ for 120 min followed by reoxygenation for 20 min in the other groups.The cells were incubated with 2.5 ％ sevoflurane for 20 min before anoxia in groups S1,S1 + SB,S3 and S3 + SB and in addition SB203580 (specific p38MAPK inhibitor) 5 μmol/L was added simultaneously in groups S1 + SB and S3 + SB.The cells were incubated with 2.5％ sevoflurane for 20 min after beginning of reoxygenation in groups S2,S2-SB,S3 and S3 + SB,and in addition SB203580 5 μmol/L was added simultaneously in groups S2 + SB and S3 + SB.The cells were incubated with SB203580 5 μmol/L for 20 min before anoxia and after beginning of reoxygenation in group SB.The lactate dehydrogenase (LDH) activity,cell survival rate and apoptotic rate were measured at the end of reoxygenation.The levels of phosphor-p38MAPK (p-p38MAPK) was detected at the end of Spre and Spost.Results Compared with group C,the LDH activity and apoptotic rate were significantly increased,while the cell survival rate was significantly decreased in the other groups (P ＜ 0.05).Compared with group A/R,the LDH activity and apoptotic rate were significantly decreased,while the cell survival rate was significantly increased in groups S1,S2 and S3 (P ＜ 0.05).There was no significant difference in the LDH activity,cell survival rate and apoptotic rate between groups S1,S2and S3 (P ＞ 0.05).The myocardial protective effect of Spre or Spost alone or in combination was eliminated by SB203580 (P ＜ 0.05).Spre or Spost alone up-regulated the expression of p-p38MAPK,Spre combined with Spost offered no additional benefit over Spre or Spost alone,and the up-regulative effect was eliminated by SB203580 (P ＜ 0.05).Conclusion Spre combined with Spost produces similar myocardial protective effect with that of either alone and it may because that both Spre and Spost attenuate A/R-induced injury to cardiomyocytes through p38MAPK signaling pathway.

Objective:To evaluate the effect of hydrogen on lipopolysaccharide (LPS)-caused inflammatory responses in BV-2 microglia and the role of autophagy.Methods:The BV-2 microglial cells cultured in vitro were seeded in 6- or 96-well plates and were divided into 4 groups ( n=24 each) using a random number table method: control group (group C), group LPS, hydrogen-rich medium group (group H) and autophagy inhibitor 3-methylpurine group (group 3-MA). In group C, cells were cultured in MEM culture medium supplemented with 15% fetal bovine serum for 24 h. In group LPS, LPS was added at a final concentration of 1 μg/ml, and cells were incubated for 24 h. In group H, LPS was added at a final concentration of 1 μg/ml, the culture medium was replaced with a hydrogen-rich medium at a final concentration of 0.6 mmol/L, and cells were incubated for 24 h. In group 3-MA, 3-methylpurine was added at a final concentration of 2 mmol/L, and the subsequent treatment was similar to those previously described in group H. The cell survival rate was detected by CCK-8 assay.The concentrations of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-10 and transforming growth factor-β (TGF-β) in supernatant were detected by enzyme-linked immunosorbent assay.The percentage of ionized calcium binding adaptor molecule-1 (Iba-1) +, Iba-1 + CD86 + and Iba-1 + CD206 + cells was detected by flow cytometry.The expression of microtubule-associated protein 1 light chain 3 Ⅰ (LC3 Ⅰ), LC3Ⅱ, Beclin-1 and p62 was detected by Western blot, and the ratio of LC3Ⅱ/LC3Ⅰ was calculated. Results:There was no significant difference in the cell survival rate among the four groups ( P>0.05). Compared with group C, the concentrations of TNF-α, IL-6, IL-10 and TGF-β and percentage of Iba-1 +, Iba-1 + CD86 + and Iba-1 + CD206 + cells were significantly increased in LPS, H and 3-MA groups, the LC3Ⅱ/LC3Ⅰ ratio and Beclin-1 expression was significantly down-regulated, and p62 expression was up-regulated in LPS and 3-MA groups, and the ratio of LC3LC3Ⅱ/LC3Ⅰ and Beclin-1 expression was significantly up-regulated, and p62 expression was down-regulated in group H ( P<0.05). Compared with group LPS, the concentrations of TNF-α and IL-6 were significantly decreased, the concentrations of IL-10 and TGF-β were increased, the percentage of Iba-1 + and Iba-1 + CD86 + cells were decreased, the percentage of Iba-1 + CD206 + cells was increased, the LC3Ⅱ/LC3Ⅰ ratio and Beclin-1 expression was up-regulated, and p62 expression was down-regulated in group H ( P<0.05), and no significant change was found in the above indexes in group 3-MA ( P>0.05). Compared with group H, the concentrations of TNF-α and IL-6 were significantly increased, the concentrations of IL-10 and TGF-β were decreased, the percentage of Iba-1 + and Iba-1 + CD86 + cells was increased, the percentage of Iba-1 + CD206 + cells was decreased, the LC3Ⅱ/LC3Ⅰ ratio and Beclin-1 expression was down-regulated, and p62 expression was up-regulated in group 3-MA ( P<0.05). Conclusion:The mechanism by which hydrogen reduces LPS-caused inflammatory responses in BV-2 microglia is related to enhancing autophagy and inhibiting microglial activation.

Objective To investigate the relationship between the mechanism underlying hydrogeninduced reduction of sepsis-associated encephalopathy (SAE) and phenotypic transformation of hippocampal microglias in mice.Methods Eighty-eight adult male ICR mice,aged 6-8 weeks,weighing 20-25 g,were divided into 4 groups (n =22 each) using a random number table method:sham operation group (group Sham),sham operation plus hydrogen group (group Sham+H2),SAE group and SAE plus hydrogen group (group SAE + H2).Sepsis was induced by cecal ligation and puncture (CLP) in anesthetized mice.Sham and Sham+H2 groups only underwent simple laparotomy.Sham+H2 and SAE+H2 groups inhaled air containing 2％ hydrogen for 1 h starting from 1 and 6 h after CLP,respectively.Mice were sacrificed at 24 h after CLP,and hippocampi were isolated for determination of the levels of tumor necrosis factor (TNF-α),interleukin-6 (IL-6),transforming growth factor-β (TGF-β) and IL-10 (by enzyme-linked immunosorbent assay) and expression of inducible nitric oxide synthase (iNOS) and argininase-1 (Arg-1) (by Western blot).Morris water maze test was performed on 10 mice in each group at days 4-8 after CLP.PResults Compared with group Sham,the levels of TNF-α,IL-6,TGF-β and IL-10 were significantly increased,the expression of iNOS and Arg-1 was up-regulated,the escape latency was prolonged,and the rate of time spent in the target quadrant and the number of crossing the original platform were reduced in SAE and SAE+H2 groups (P＜0.05).Compared with group SAE,the levels of TNF-α and IL-6 were significantly decreased,the expression of iNOS was down-regulated,the expression of TGF-β,IL-10 and Arg-1 was up-regulated,the escape latency was shortened,and the rate of time spent in the target quadrant and the number of crossing the original platform were increased in group SAE+H2 (P＜0.05).Conclusion Hydrogen can promote phenotypic transformation of hippocampal microglias from M1 to M2 and reduce SAE in mice.