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Objective To evaluate the effect of hydrogen on the expression of Drp1 in the myocardial mitochondria of septic mice.Methods Fifty-four pathogen-free healthy male ICR mice,aged 6 weeks,weighing 20-25 g,were divided into 3 groups (n=18 each) using a random number table:sham operation group (S group),sepsis group (Sep group) and hydrogen group (H2 group).Sepsis was produced by cecal ligation and puncture in anesthetized mice.The mice inhaled 2％ hydrogen for 1 h starting from 1 and 6 h after operation in group H2.The mice were sacrificed at 1 day after operation,and hearts were removed for microscopic examination of the pathological changes which were scored and for determination of apoptosis in cardiomyocytes.Apoptosis rate was calculated.The myocardial mitochondria were isolated for measurement of respiratory control ratio (RCR) (using a Clark-type electrode),ATP content (by using a bioluminescence technique) and Drp1 expression (by Western blot).Results Compared with group S,the pathological score and apoptosis rate were significantly increased,the RCR and ATP content in mitochondria were decreased,and the expression of Drp1 was up-regulated in Sep and H2 groups (P＜0.05).Compared with group Sep,the pathological score and apoptosis rate were significantly decreased,the RCR and ATP content in mitochondria were increased,and the expression of Drp1 was down-regulated in group H2 (P＜0.05).Conclusion The mechanism by which hydrogen mitigates myocardial injury is related to down-regulating expression of Drp1 in the myocardial mitochondria and improving mitochondrial function in septic mice.
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Objective To evaluate the role of autophagy in hydrogen-induced reduction of lung injury in septic mice.Methods Sixty pathogen-free healthy male ICR mice,aged 6 weeks,weighing 20-25 g,were divided into 5 groups (n =12 each) using a random number table:sham operation group (group Sh),sepsis group (group Sep),sepsis plus hydrogen group (group Sep+H2),sepsis plus autophagy inhibitor 3-methyladenine (3-MA) group (group Sep+3-MA) and sepsis plus 3-MA plus hydrogen group (group Sep+3-MA+H2).Sepsis was produced by cecal ligation and puncture.At 1 h before operation,3-MA 10 mg/kg was intraperitoneally injected.The mice inhaled 2％ H2 for 1 h starting from 1 and 6 h after operation.Blood samples were collected from the common carotid artery at 24 h after operation for measurement of arterial oxygen partial pressure,and the oxygenation index (OI) was calculated.Pulmonary specimens were obtained for examination of the pathological changes which were scored.Pulnonary mitochondria were isolated for determination of mitochondrial membrane potential (MMP) and ATP content using fluorescence spectrophotometry and a bioluminescence assay,respectively,and the respiratory control rate (RCR) was calculated.The expression of autophagy-related protein microtubule-associated protein 1 light chain 3 (LC3) was determined by Western blot,and the ratio of LC3-Ⅱ to LC3-Ⅰ expression (LC3-Ⅱ/LC3-Ⅰ ratio) was calculated.Results Compared with group Sh,the pathological scores were significantly increased,the OI and contents of mitochondrial RCR,MMP and ATP were decreased,and the LC3-Ⅱ/LC3-Ⅰ ratio was increased in Sep and Sep+H2 groups (P＜0.05).Compared with group Sep,the pathological scores were significantly decreased,the OI and contents of mitochondrial RCR,MMP and ATP were increased,and the LC3-Ⅱ/LC3-Ⅰ ratio was increased in group Sep+H2,and the pathological scores were significantly increased,the OI and contents of mitochondrial RCR,MMP and ATP were decreased,and the LC3-Ⅱ/LC3-Ⅰ ratio was decreased in group Sep+3-MA (P＜0.05),and no significant change was found in each parameter mentioned above in group Sep+3-MA+H2 (P＞0.05).Compared with group Sep+H2,the pathological scores were significantly increased,the OI and contents of mitochondrial RCR,MMP and ATP were decreased,and the LC3-Ⅱ/LC3-Ⅰ ratio was decreased in group Sep+3-MA+H2 (P＜0.05).Conclusion The mechanism by which hydrogen ameliorates lung injury is related to enhanced level of autophagy in septic mice.
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Objective To evaluate the effect of hydrogen on mitochondrial dynamics during endotoxin-induced damage to human umbilical vein endothelial cells (HUVECs).Methods HUVECs cultured in vitro were seeded in the culture plate and divided into 4 groups using a random number table:control group (group C),hydrogen-saturated culture medium group (H group),endotoxin group (group E) and endotoxin + hydrogen-saturated culture medium group (group E+H).The cells were cultured in the plain culture medium in C and E groups.The cells were cultured in the hydrogen-saturated culture medium containing lipopolysaccharide (LPS) with the final concentration of 10 μg/ml in H and E+H groups.At 2,8 and 24 h of culture or incubation with LPS,the cell viability was detected by methyl thiazolyl tetrazolium assay,the intracellular ATP content was measured using the phosphomolybdic acid colorimetric method,and the expression of dynamin-related protein 1 (DRP1) was detected by using Western blot.The expression of DRP1 was detected by immunofluorescence at 8 h of incubation with LPS.Results Compared with group C,the cell viability and ATP content were significantly decreased,and the expression of DRP1 was up-regulated at each incubation time point in E and E +H groups (P＜0.05),and no significant change was found in the parameters mentioned above in group H (P＞0.05).Compared with group E,the cell viability and ATP content were significantly increased,and the expression of DRP1 was down-regulated at each incubation time point in group E+H (P＜0.05).Conclusion The mechanism by which hydrogen reduces endotoxin-induced damage to HUVECs is related to down-regulation of DRP1 expression and inhibition of excessive mitochondrial fission.
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Objective To evaluate the effect of inhaling hydrogen (H2) on proteomics during acute lung injury in septic mice.Methods Sixty male ICR mice,aged 6 weeks,weighing 20-25 g,were divided into 4 groups (n=15 each) using a random number table:sham operation group (Sh group),sham operation plus H2 group (Sh+H2 group),sepsis group (S group) and sepsis plus H2 group (S+H2 group).Sepsis was produced by cecal ligation and puncture.The mice in Sh+H2 and S+H2 groups inhaled 2％ H2 for 1 h starting from 1 and 6 h after operation.At 24 h after operation,lungs were removed for identification of proteins by isobaric tags for relative and absolute quantification and liquid chromatography-tandem mass spectrometry analysis,and the differentially expressed proteins were screened.The differentially expressed proteins were used for KEGG pathway enrichment analysis and STRING protein-protein interaction networks analysis.Western blot was used to confirm the 4 differentially expressed proteins semaphorin 7A,transferrin,OTULIN and mitogen-activated protein kinase kinase kinase 1.Results A total of 4 472 quantifiable proteins were identified.A total of 192 proteins which were related to acute lung injury during H2 inhalationinduced reduction of sepsis were identified.The 192 proteins involved phosphatidylinositol 3-kinase/serine0threonine kinase signaling pathway,chemokine signaling pathway,hypoxia-inducible factor 1 signaling pathway,complement and coagulation cascades,peroxisome proliferator-activated receptor signaling pathway and proteins including ribosome proteins,myosin and troponin,collagen and adhesion-related proteins,coagulation-related proteins found in STRING protein-protein interaction networks.Conclusion Inhaling H2 can induce changes in the expression of 192 proteins,which may be the mechanism of lung protection in septic mice.
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Objective To evaluate the relationship between the endogenous protection induced by autophagy against acute lung injury and inflammatory responses in the septic mice.Methods A total of 130 pathogen-free male C57BL/6 mice,aged 6 weeks,weighing 20-25 g,were randomly divided into 5 groups (n =26 each) using a random number table:sham operation group (group S),sepsis group (group Sep),autophagy inducer rapamycin group (group Rap),autophagy inhibitor 3-methyladenine (3-MA) group (group 3-MA),and lysosome inhibitor bafilomycin group (group Baf).Sepsis was produced by cecal ligation and puncture in chloral hydrate-anesthetized mice.Rapamycin 10 mg/kg,3-MA 15 mg/kg,and bafilomycin 1 mg/kg were injected intraperitoneally at 1 h after operation in Rap,3-MA and Baf groups,respectively.Twenty mice in each group were selected and observed for the survival at 7 days after operation,and the 7-day survival rate was calculated.Six mice in each group were selected,and arterial blood samples were obtained at 24 h after operation,the mice were then sacrificed,and lung tissues were obtained for determination of the levels of tumor necrosis factor-alpha (TNF-α),high-mobility group box 1 (HMGB1),interleukin-6 (IL-6),IL-10 and monocyte chemotactic protein-1 (MCP-1) in serum and lung tissues by enzyme-linked immunosorbent assay.The expression of autophagy-related proteins microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ),Beclin-1 and lysosomal-associated membrane protein 2 (LAMP2),and Ras-related protein 7 (Rab7) in lung tissues was detected by Western blot.Results Compared with group S,the 7-day survival rate was significantly decreased,and the levels of TNF-α,HMGB1,IL-6,IL-10 and MCP-1 in serum and lung tissues and LC3 Ⅱ,Beclin-1,LAMP2 and Rab7 in lung tissues were significantly increased in group Sep (P＜0.05).Compared with group Sep,the 7-day survival rate and levels of IL-10,LAMP2 and Rab7 in serum and lung tissues were significantly increased in group Rap and decreased in 3-MA and Baf groups,and the levels of TNF-α,HMGB1,IL-6 and MCP1 in serum and lung tissues were significantly decreased in group Rap and increased in 3-MA and Baf groups,and the levels of LC3 Ⅱ and Beclin-1 in lung tissues were significantly increased in Rap and Baf groups and decreased in group 3-MA (P＜0.05).Conclusion Autophagy-induced endogenous protection against acute lung injury is related to inhibition of inflammatory responses in the septic mice.
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Objective To evaluate the effect of hydrogen ( H2 ) inhalation on mitochondrial bio?synthesis in lung tissues during acute lung injury in mice with sepsis. Methods One hundred and four male ICR mice, aged 6 weeks, weighing 20-25 g, were divided into 4 groups ( n=26 each) using a ran?dom number table: sham operation group ( group S) , sham operation + H2 group ( group S+H2 ) , sepsis group ( group Sep) and sepsis + H2 group ( group Sep+H2 ) . Sepsis was produced by cecal ligation and puncture. In S+H2 and Sep+H2 groups, the mice inhaled 2% H2 for 1 h starting from 1 and 6 h after opera?tion. Twenty mice in each group were selected, and the survival rates on postoperative days 1, 2, 3, 5 and 7 were recorded. On the postoperative day 1, 6 mice in each group were selected, and blood samples were collected from the common carotid artery for measurement of arterial oxygen partial pressure, and the oxygenation index was calculated. The pulmonary specimens were obtained for examination of the pathologi?cal changes which were scored and for determination of the expression of peroxisome proliferator?activated re?ceptor gamma coactivator?1α ( PGC?1α) in lung tissues by Western blot. The pulmonary mitochondria were isolated for determination of mitochndrial membrane potential ( MMP ) and ATP contents using spectropho?tometry and a bioluminescence technique, respectively. Results Compared with group S, the survival rate, oxygenation index and MMP and ATP content in lung tissues were significantly decreased, and the pathological scores and PGC?1α expression in lung tissues were significantly increased in Sep and Sep+H2 groups (P<0.05). Compared with group Sep, the survival rate, oxygenation index, and MMP, ATP content and PGC?1α expression in lung tissues were significantly increased, and the pathological scores were significantly decreased in group Sep+H2 ( P<0.05) . Conclusion H2 inhalation can ameliorate acute lung injury in mice with sepsis, and the mechanism is associated with the enhanced function of PGC?1αand promoted mitochondrial biosynthesis in lung tissues.
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Objective To investigate the role of autophagy in the lung injury in the septic mice.Methods Thirty-six male C57BL/6 mice, aged 6 weeks, weighing 20-25 g, were randomly divided into 3 groups (n=12 each) using a random number table: sham operation group (group S);cecal ligation and puncture (CLP) group;CLP + autophagy inhibitor 3-methyladenine (3-MA) group (group CLP+3-MA).Sepsis was produced by CLP.In group CLP+3-MA, 3-MA 10 mg/kg was injected intraperitoneal at 1 h after operation.Arterial blood samples were taken at 24 h after operation for blood gas analysis, and the oxygenation index was calculated.The lungs were removed for microscopic examination of pathologic changes which were scored, and for determination of wet/dry lung weight ratio (W/D ratio) , myeloperoxidase (MPO) activity (using colorimetric method) and the expression of autophagy protein microtubule-associated protein 1 light chain 3 Ⅱ] (LC3 Ⅱ), Beclin-1 and lysosomes-associated protein Rab7 and lysosome-associated membrane protein-2 (LAMP2) (by Western blot).The lung was lavaged, and broncho-alveolar lavage fluid (BALF) was collected for determination of the total cell count and polymorphonuclear leukocyte (PMN) count.Results Compared with group S, the pathological score, W/D ratio, MPO activity, and the total cell and PMN counts in BALF were significantly increased, and oxygenation index was decreased in CLP and CLP +3-MA groups, and the expression of LC3 Ⅱ , Beclin-1, LAMP2 and Rab7 was up-regulated in group CLP (P＜0.05).Compared with group CLP, the pathological score, W/D ratio, MPO activity, and the total cell and PMN counts in BALF were significantly increased, the oxygenation index was decreased, and the expression of LC3 Ⅱ , Beclin-1, LAMP2 and Rab7 was down-regulated in group CLP+ 3-MA (P＜0.05).Conclusion Autophagy is involved in the endogenous protective mechanism of acute lung injury in the septic mice.