Objective:To evaluate the relationship between phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) signaling pathway and heme oxygenase-1 (HO-1) expression during lung injury in septic mice.Methods:Forty-eight clean-grade 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 SH), sepsis group (group SEP), PI3K inhibitor LY294002 group (group LY) and LY294002+ HO-1 agonist hemin group (group LH). Sepsis was induced by cecal ligation and puncture in anesthetized animals.LY294002 30 mg/kg was intraperitoneally injected at 2 h before establishing the model in LY group and LH group.Hemin 50 μmol/kg was intraperitoneally injected at 1 h before establishing the model in LH group.Mice were sacrificed at 24 h after surgery, and lungs were removed for determination of wet/dry weight ratio (W/D ratio), tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) contents (by enzyme-linked immunosorbent assay), and expression of phosphorylated Akt(p-Akt), Akt and HO-1 (by Western blot) and for examination of the pathological changes of lung tissues which were scored. Results:Compared with SH group, the W/D ratio, lung injury score, and TNF-α and IL-10 contents were significantly increased in SEP, LY and LH groups, and the expression of p-Akt and HO-1 was significantly up-regulated in SEP group ( P<0.05). Compared with SEP group, the W/D ratio, lung injury score and TNF-α content were significantly increased, IL-10 content was decreased, and the expression of p-Akt and HO-1 was down-regulated in LY group ( P<0.05). Compared with LY group, the lung injury score and TNF-α content were significantly decreased, IL-10 content was increased, and the expression of HO-1 was up-regulated in LH group ( P<0.05). Conclusion:PI3K/Akt signaling pathway is involved in the endogenous protective mechanism of lung injury by regulating HO-1 expression in septic mice.

Objective To evaluate the role of mitophagy in hydrogen-induced reduction of lipopolysaccharide (LPS)-caused mitochondrial injury to macrophages of mice.Methods Macrophage line RAW264.7 cells of mice were routinely cultured and divided into 4 groups (n =6 each) using a random number table method:control group (Con group),LPS group,LPS plus hydrogen group (LPS + H2 group) and LPS plus hydrogen plus mitophagy inhibitor 3-methyladenine (3-MA) group (LPS+H2+3-MA group).Cells were incubated for 6 h with LPS at the concentration of 1 μg/ml in LPS group.Cells were incubated for 6 h with LPS 1 μg/ml and hydrogen-rich medium 0.6 mmol/L.Cells were incubated for 1 h with 2 mmol/L 3-MA and then incubated for 6 h with LPS 1 μg/ml and hydrogen-rich medium 0.6 mmol/L in LPS+H2+3-MA group.Mitochondrial respiratory control ratio (RCR) was measured using a Clark-type electrode.Mitochondrial membrane potential (MMP) was determined by JC-1 staining.Autophagosomes were counted with a transmission electron microscope.The expression of PTEN-induced putative kinase 1 (PINK1),E3 ubiquitin ligase (Parkin),microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ)and Beclin-1 was determined by Western blot.Results Compared with Con group,RCR and MMP were significantly decreased,the expression of PINK1,Parkin,LC3 Ⅱ and Beclin-1 was up-regulated,and the number of autophagosomes was increased in LPS group (P＜0.05).Compared with LPS group,RCR and MMP were significantly increased,the expression of PINK1,Parkin,LC3 Ⅱ and Beclin-1 was up-regulated,and the number of autophagosomes was increased in LPS+H2group (P＜0.05).Compared with LPS+H2 group,RCR and MMP were significantly decreased,the expression of PINK1,Parkin,LC3 Ⅱ and Beclin-1 was down-regulated,and the number of autophagosomes was decreased in LPS + H2 + 3-MA group (P＜0.05).Conclusion Enhanced mitophagy is involved in hydrogen-induced reduction of LPS-caused mitochondirial injury to macrophages of mice.

Objective: To evaluate the effect of hydrogen on the mitochondrial function in brain tissues of mice with sepsis-associated encephalopathy (SAE). Methods: Two hundred and sixty-eight healthy male C57 mice, aged 6 weeks, weighing 20-25 g, were divided into 4 groups (n=67 each) using a random number table method: sham operation group (group SH), sham operation plus hydrogen gas group (group SH+ H₂), group SAE, and SAE plus hydrogen gas group (group SAE+ H₂). Sepsis was produced by cecal ligation and puncture in anesthetized mice.The mice in group SH+ H₂ and group SAE+ H₂ inhaled 2% hydrogen gas for 1 h starting from 1 and 6 h after operation, respectively.The postoperative 7-day survival rate was recorded.Brain tissues were obtained at 24 h after operation to count the normal neurons in hippocampal CA1 region.At 6, 12 and 24 h after operation, hippocampal mitochondria were isolated for determination of mitochondrial membrane potential (MMP) by fluorescence spectrophotometry and ATP content by a bioluminescence assay.Y-maze (spontaneous alternation) test was performed at days 3, 5 and 7 after operation. Results: Compared with group SH, the 7-day survival rate was significantly decreased, the number of normal neurons in hippocampal CA1 region, MMP, mitochondrial ATP content and spontaneous alternation percentage in Y-maze test were significantly decreased in group SAE and group SAE+ H₂ (P<0.05). Compared with group SAE, the 7-day survival rate, the number of normal neurons in hippocampal CA1 region, MMP, mitochondrial ATP content and spontaneous alternation percentage in Y-maze test were significantly increased in group SAE+ H₂ (P<0.05). Conclusion The mechanism by which hydrogen reduces SAE is probably associated with improving mitochondrial function in brain tissues of mice.

Objective: To evaluate the effect of hydrogen on mitochondrial biosynthesis in the hippocampus of mice with sepsis-associated encephalopathy (SAE). Method: Two hundred and twenty-four healthy clean-grade male C57BL/6J mice, aged 6-8 weeks, weighing 20-25 g, were divided into 4 groups (n=56 each) using a random number table method: sham operation group (group SH), sham operation plus hydrogen group (group SH+ H₂), group SAE, and SAE plus hydrogen group (group SAE+ H₂). The model of SAE was established by cecal ligation and puncture in anesthetized mice.Group SH+ H₂ and group SAE+ H₂ inhaled 2% hydrogen for 1 h starting from 1 and 6 h after operation, respectively.Twenty mice were selectde to record the postoperative 7-day survival rate.The remaining animals were sacrificed at 24 h after operation, and brain tissues were taken for examination of the pathological changes in hippocampal CA1 region (with a light microscope) and for determination of neuronal apoptosis (by TUNEL), mitochondrial membrane potential (MMP) (by fluorescence spectrophotometry) and ATP content (by a bioluminescence assay). The apoptosis rate was calculated.The expression of peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-1α) in hippocampus was determined by Western blot at 6, 12 and 24 h after operation. Results: Compared with group SH, the postoperative 7-day survival rate was significantly decreased, the apoptosis rate of hippocampal neurons was increased, the contents of MMP and ATP were decreased, and the expression of PGC-1α was up-regulated in SAE and SAE+ H₂groups (P<0.05), and no significant change was found in the parameters mentioned above in group SH+ H₂(P>0.05). Compared with group SAE, the postoperative 7-day survival rate was significantly increased, and the apoptosis rate of hippocampal neurons was decreased, the contents of MMP and ATP were increased, and the expression of PGC-1α was up-regulated (P<0.05), and the pathological changes of hippocampal tissues were significantly attenuated in group SAE+ H₂. Conclusion The mechanism by which hydrogen mitigates SAE may be related to promoting mitochondrial biosynthesis in mice.

OBJECTIVE： To study the effects of glycyrrhizic acid on the pharmacokinetics of nifedipine in rats. METHODS： Rats were randomly divided into experimental group and control group， with 10 rats in each group. Experimental group was given glycyrrhizic acid 5 mg/kg and control group was given 0.5％ CMC-Na （sodium carboxymethylcellulose） solution， once a day， for 14 consecutive days. On 14th day after 30 min of intragastric administration， both groups were given nifedipine 3 mg/kg intragastrically. Blood samples 0.5 mL were collected from intraocular vein plexus before and at 0.25， 0.5， 0.75， 1， 1.5， 2， 3， 4， 6， 8， 12 and 24 h after intragastric administration. The concentration of nifedipine was determined by HPLC using diazepam as internal standard. The determination was performed on ODS-C18 column with mobile phase consisted of methanol-water （62 ∶ 38，     V/V，pH adjusted to 4.5 with acetic acid） at the flow rate of 1.0 mL/min. The column temperature was 30 ℃. The detection wavelength was set at 238 nm， and sample size was 20 μL. The pharmacokinetic parameters were calculated with Winonlin 6.0 software， and statistical analysis was performed by t-test. RESULTS： The main pharmacokinetic parameters of the experimental group and the control group were as follows as tmax was （1.40±0.15）， （1.50±0.01） h； cmax was （0.15±0.03）， （0.29±0.09）      mg/L； t1/2 was （4.70±1.17）， （5.20±1.38） h； AUC0-24 h were （1.00±0.10）， （1.89±0.37） mg·h/L； AUC0-∞ was （1.00±0.16）， （1.98±0.32） mg·h/L； MRT was （6.76±0.64）， （6.60±1.36） h， respectively. Compared with control group， cmax， AUC0-24 h and AUC0-∞ of nifedipine were decreased significantly in experimental group， with statistical significance （P＜0.05）. CONCLUSIONS： Glycyrrhizic acid can reduce the bioavailability of nifedipine in rats. It is suggested that the dosage of nifedipine should be increased in order to achieve effective blood concentration.

Objective To evaluate the effect of hydrogen on the mitochondrial function in brain tis-sues of mice with sepsis-associated encephalopathy(SAE).Methods Two hundred and sixty-eight healthy male C57 mice,aged 6 weeks,weighing 20-25 g,were divided into 4 groups(n＝67 each)using a ran-dom number table method: sham operation group(group SH),sham operation plus hydrogen gas group(group SH+H2),group SAE,and SAE plus hydrogen gas group(group SAE+H2).Sepsis was produced by cecal ligation and puncture in anesthetized mice.The mice in group SH+H2 and group SAE+H2 inhaled 2％hydrogen gas for 1 h starting from 1 and 6 h after operation,respectively.The postoperative 7-day sur-vival rate was recorded.Brain tissues were obtained at 24 h after operation to count the normal neurons in hippocampal CA1 region.At 6,12 and 24 h after operation,hippocampal mitochondria were isolated for determination of mitochondrial membrane potential(MMP)by fluorescence spectrophotometry and ATP con-tent by a bioluminescence assay.Y-maze(spontaneous alternation)test was performed at days 3,5 and 7 after operation.Results Compared with group SH,the 7-day survival rate was significantly decreased,the number of normal neurons in hippocampal CA1 region,MMP,mitochondrial ATP content and sponta-neous alternation percentage in Y-maze test were significantly decreased in group SAE and group SAE+H2(P＜0.05).Compared with group SAE,the 7-day survival rate,the number of normal neurons in hipp-ocampal CA1 region,MMP,mitochondrial ATP content and spontaneous alternation percentage in Y-maze test were significantly increased in group SAE+H2(P＜0.05).Conclusion The mechanism by which hy-drogen reduces SAE is probably associated with improving mitochondrial function in brain tissues of mice.

Objective To evaluate the effect of dexmedetomidine on expression of hypoxia-inducible factor-1α (HIF-1α) during endotoxin-caused apoptosis in macrophages of mice.Methods Mouse macrophage cell line RAW264.7 cultured in vitro were seeded in 6-well or 96-well plates and divided into 4 groups (n=16 each) when cell confluence reached 60％-70％ using a random number table method:control group (group Con),dexmedetomidine group (group Dex),lipopolysaccharide (LPS) group,and LPS plus dexmedetomidine group (group LPS+Dex).Phosphate buffer solution was added in group Con.Dexmedetomidine 1 μmol/L was added in group Dex.LPS 1 μg/ml was added in LPS and LPS+Dex groups.Dexmedetomidine 1 μmol/L was added immediately after adding LPS in group LPS+Dex.Cells were then cultured for 24 h in each group.Cell apoptosis was measured using TUNEL,mitochondrial membrane potential using JC-1,reactive oxygen species (ROS) content by ROS kit,and ATP content by ATP kit.The apoptosis rate was calculated.The expression of HIF-1α,cytochrome C (Cyt-c),caspase-9 and cleaved caspase-3 was detected by Western blot.Results Compared with group Con,the apoptosis rate and ROS content were significantly increased,ATP content and mitochondrial membrane potential were decreased,the expression of HIF-1α,Cyt-c,caspase-9 and cleaved caspase-3 was up-regulated in group LPS (P＜ 0.05),and no significant change was found in the parameters mentioned above in group Dex (P＞0.05).Compared with group LPS,the apoptosis rate and ROS content were significantly decreased,ATP content and mitochondrial membrane potential were increased,the expression of HIF-1α was up-regulated,and the expression of Cyt-c,caspase-9 and cleaved caspase-3 was down-regulated in group LPS + Dex (P＜0.05).Conclusion Dexmedetomidine can reduce endotoxin-caused oxidative stress injury to macrophages,improve mitochondrial function and inhibit mitochondrial apoptosis,and the mechanism may be related to upregulating the expression of HIF-1α in mice.

Objective To evaluate the role of autophagy in dexmedetomidine-induced reduction of lipopolysaccharide ( LPS)-caused inflammatory responses in macrophages of mice. Methods Mouse mac-rophage cell line RAW264. 7 cultured in vitro were seeded in 6-well or 96-well plates and divided into 4 groups ( n=20 each ) when cell confluence reached 60％ using a random number table method: control group (group Con), LPS group, LPS plus dexmedetomidine group (group LPS+DEX), and LPS plus dexmedetomidine plus autophagy inhibitor 3-MA group (group LPS+DEX+3-MA). PBS was added and cells were cultured for 12 h in group Con. LPS at the final concentration of 1000 ng∕ml was added and cells were incubated for 12 h in group LPS. LPS at the final concentration of 1000 ng∕ml was added, and then dexmedetomidine at the final concentration of 1 μmol∕L was immediately added, and cells were incubated for 12 h in group LPS+Dex. In group LPS+Dex+3-MA, 3-MA at the final concentration of 2 mmol∕L was added and cells were incubated for 1 h, LPS at the final concentration of 1000 ng∕ml was added, and then dexmedetomidine at the final concentration of 1 μmol∕L was immediately added, and cells were incubated for 12 h. Cell viability was detected by CCK-8 assay, and the concentrations of nitrous oxide ( NO) , tumor necrosis factor-alpha ( TNF-α) and interleukin-1beta ( IL-1β) in the supernatant were determined by en-zyme-linked immunosorbent assay, and the expression of microtubule-associated protein 1 light chain 3 Ⅰ( LC3 Ⅰ) , LC3Ⅱ, P62 and Bcelin-1 was detected by Western blot. LC3Ⅱ∕LC3Ⅰ ratio was calculated. Results Compared with group Con, the cell viability was significantly decreased, the concentrations of TNF-α, IL-1β and NO and LC3Ⅱ∕LC3Ⅰratio were increased, and the expression of P62 and Beclin1 was up-regulated in group LPS (P<0. 05). Compared with group LPS, the cell viability was significantly in-creased, the concentrations of TNF-α, IL-1βand NO were decreased, LC3Ⅱ∕LC3Ⅰratio was increased, the expression of P62 was down-regulated, and the expression of Beclin1 was up-regulated in group LPS+DEX ( P<0. 05) . Compared with group LPS+Dex, the cell viability was significantly decreased, the con-centrations of TNF-α, IL-1β and NO were increased, LC3Ⅱ∕LC3Ⅰ ratio was decreased, the expression of P62 was up-regulated, and the expression of Beclin1 was down-regulated in group LPS+Dex+3-MA ( P<0. 05) . Conclusion Enhanced autophagy is involved in dexmedetomidine-induced reduction of LPS-caused inflammatory responses in macrophages of mice.

Objective To investigate the role of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome in hydrogen-rich saline-induced reduction of lipopolysaccharide (LPS)-caused damage to mitochondria in macrophages of mice.Methods Macrophage line RAW264.7 of mice were routinely cultured and divided into 4 groups (n=6 each) using a random number table method:control group (group C),group LPS,hydrogen-rich saline plus LPS group (group LPS+H2) and hydrogen-rich saline plus LPS plus ATP group (group LPS+ATP+H2).LPS was given at the concentration of 1 μg/ml,and the cells were then incubated for 30 min in group LPS.LPS at the concentration of 1 μg/ml and hydrogen-rich saline at the concentration of 0.6 mmol/L were simultaneously given,and the cells were then incubated for 30 min in LPS+H2 and LPS+ATP+H2 groups.ATP at the concentration of 1 nmol/L was then given,and the cells were incubated for 6 h in group LPS+ATP+H2.Mitochondrial membrane potential (MMP) was determined by JC-1 staining,and respiratory control ratio (RCR) was measured using a Clark-type electrode.The expression of NLRP3,caspase-1 and apoptosisassociated speck-like protein containing C-terminal caspase recruitment domain (ASC) was determined by Western blot.The concentrations of INTERLEUKIN-1 BETA (IL-1β),IL-18 and IL-6 in the supernatant were determined by enzyme-linked immunosorbent assay.Results Compared with group C,MMP and RCR were significantly decreased,the concentrations of IL-1β,IL-18 and IL-6 in the supernatant were increased,and the expression of NLRP3,caspase-1 and ASC was up-regulated in group LPS (P＜0.05).Compared with group LPS,MMP and RCR were significantly increased,the concentrations of IL-1β,IL-l8 and IL-6 in the supernatant were decreased,and the expression of NLRP3,caspase-1 and ASC was down-regulated in group LPS+H2 (P＜0.05).Compared with group LPS+H2,MMP and RCR were significantly increased,the concentrations of IL-1β,IL-18 and IL-6 in the supernatant were decreased,and the expression of NLRP3,caspase-1 and ASC was down-regulated in group LPS+ATP+H2 (P＜0.05).Conclusion Hydrogen-rich saline can reduce LPS-caused damage to mitochondria in macrophages of mice through inhibiting the activation of NLRP3 inflammasome.

Objective To evaluate the relationship between phosphatidylinositol 3?kinase∕serine?threonine kinase(PI3K∕Akt)signaling pathway and autophagy during acute lung injury in septic mice. Methods Thirty?six male C57BL∕6 mice, aged 6-8 weeks, weighing 20-25 g, were divided into 3 groups(n=12 each)using a random number table: sham operation group(group SH), sepsis group (group S)and PI3K inhibitor LY294002 plus sepsis group(group LY+S). Sepsis was induced by cecal ligation and puncture in S and LY+S groups. LY294002 30 mg∕kg was intraperitoneally injected at 2 h be?fore operation in group LY+S. Arterial blood samples were taken at 24 h after operation for blood gas analy?sis, PaO2was recorded, and oxygenation index was calculated. Lung specimens were obtained for examina?tion of pathological changes which were scored and for determination of autophagosome count(using trans?mission electron microscope), wet∕dry weight ratio(W∕D ratio)and expression of Akt, phosphorylated Akt(p?Akt), Beclin?1 and microtubule?associated protein 1 light chain 3 Ⅱ(LC3 Ⅱ). The p?Akt∕Akt ratio was calculated. Results Compared with group SH, oxygenation index was significantly decreased,and the W∕D ratio and pathological score were increased in S and LY+S groups, the autophagosome count was significantly increased, p?Akt∕Akt ratio was increased, and the expression of Beclin?1 and LC3Ⅱ was up?regulated in group S(P<0.05). Compared with group S, oxygenation index was significantly de?creased, and the W∕D ratio was increased, the autophagosome count was decreased, pathological scores were increased, p?Akt∕Akt ratio was decreased, and the expression of Beclin?1 and LC3Ⅱwas down?regu?lated in group LY+S(P<0.05). Conclusion PI3K∕Akt signaling pathway activation mediates autophagy and is involved in the endogenous protective mechanism of acute lung injury in septic mice.