Aim To investigate the effect that catalpol intervenes macrophage polarization mediated by mouse mesangial cells(MMCs) stimulated by advanced glycation end products(AGEs).Methods RAW264.7 macrophages and MMCs were co-cultured in vitro and divided into model group(100 mg·L-1 AGEs), control group(100 mg·L-1 BSA), catalpol(0.1, 1.0, 10.0 μmol·L-1) group, and aminoguanidine(1.0 μmol·L-1) group which was set as positive control.After being incubated with catalpol for 1 h, MMCs were stimulated by AGEs for 23 h.The proliferation-inhibition rate of MMCs was measured by MTT assay.MCP-1 in supernatant liquid of MMCs was detected by ELISA method.The expression of iNOS, CD16/32, TNF-α, COX-2, CD206 and Arg-1 was detected by Western blot.Simultaneously, the percentage of iNOS and CD206 was also measured by flow cytometry.Results AGEs could increase the level of MCP-1 secreted by MMCs.The expression of iNOS, TNF-α, CD16/32 and COX-2 protein of macrophage was up-regulated after MMCs stimulated by AGEs, while the expression of CD206 and Arg-1 was down-regulated.After being intervened by catalpol, these effects could be reversed.All the changes were concentration-related.Conclusions Catalpol can inhibit macrophages M1-type polarization process and promote M2-type polarization, which may be mediated through MCP-1 secreted by MMCs after AGEs stimulation.Catalpol can ameliorate inflammation and relieve diabetic kidney injury.

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.

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.

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.

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.

Objective: To investigate the effects of hydrogen on the lung damage of mice at early stage of severe burn. Methods: One hundred and sixty ICR mice were divided into sham injury, hydrogen, pure burn, and burn+ hydrogen groups according to the random number table, with 40 mice in each group. Mice in pure burn group and burn+ hydrogen group were inflicted with 40% total body surface area full-thickness scald (hereafter referred to as burn) on the back, while mice in sham injury group and hydrogen group were sham injured. Mice in hydrogen group and burn+ hydrogen group inhaled 2% hydrogen for 1 h at post injury hour (PIH) 1 and 6, respectively, while mice in sham injury group and pure burn group inhaled air for 1 h. At PIH 24, lung tissue of six mice in each group was harvested, and then pathological changes of lung tissue were observed by HE staining and the lung tissue injury pathological score was calculated. Inferior vena cava blood and lung tissue of other eight mice in each group were obtained, and then content of high mobility group box 1 (HMGB1) and interleukin-6 (IL-6) in serum and lung tissue was determined by enzyme-linked immunosorbent assay. Activity of superoxide dismutase (SOD) in serum and lung tissue was detected by spectrophotometry. After arterial blood of other six mice in each group was collected for detection of arterial partial pressure of oxygen (PaO₂), the wet and dry weight of lung tissue were weighted to calculate lung wet to dry weight ratio. The survival rates of the other twenty mice in each group during post injury days 7 were calculated. Data were processed with one-way analysis of variance, LSD test and log-rank test. Results: (1) At PIH 24, lung tissue of mice in sham injury group and hydrogen group showed no abnormality. Mice in pure burn group were with pulmonary interstitial edema, serious rupture of alveolar capillary wall, and infiltration of a large number of inflammatory cells. Mice in burn+ hydrogen group were with mild pulmonary interstitial edema, alveolar capillary congestion accompanied by slight rupture and bleeding, and the number of infiltration of inflammatory cells was smaller than that in pure burn group. The lung tissue injury pathological scores of mice in sham injury group, hydrogen group, pure burn group, and burn+ hydrogen group were (0.7±0.5), (0.8±0.5), (6.1±1.0), and (2.8±0.8) points, respectively. The lung tissue injury pathological score of mice in pure burn group was significantly higher than that in sham injury group (P<0.001). The lung tissue injury pathological score of mice in burn+ hydrogen group was significantly lower than that in pure burn group (P<0.001). (2) At PIH 24, the content of HMGB1 and IL-6 in serum and lung tissue of mice in hydrogen group was close to that in sham injury group (with P values above 0.05). The content of HMGB1 and IL-6 in serum and lung tissue of mice in pure burn group was significantly higher than that in sham injury group (with P values below 0.001). The content of HMGB1 and IL-6 in serum and lung tissue of mice in burn+ hydrogen group was significantly lower than that in pure burn group (with P values below 0.001). (3) At PIH 24, the activity of SOD in serum and lung tissue of mice in hydrogen group was close to that in sham injury group (with P values above 0.05). The activity of SOD in serum and lung tissue of mice in pure burn group was significantly lower than that in sham injury group (with P values below 0.001). The activity of SOD in serum and lung tissue of mice in burn+ hydrogen group was significantly higher than that in pure burn group (with P values below 0.001). (4) At PIH 24, there was no statistically significant difference in PaO₂ among the mice in four groups (F=0.04, P>0.05). (5) At PIH 24, the ratios of lung wet to dry weight of mice in sham injury, hydrogen, pure burn, and burn+ hydrogen groups were 3.52±0.22, 3.61±0.24, 7.24±0.32, and 5.21±0.41, respectively. The ratio of lung wet to dry weight of mice in pure burn group was significantly higher than that in sham injury group (P<0.001). The ratio of lung wet to dry weight of mice in burn+ hydrogen group was significantly lower than that in pure burn group (P<0.001). (6) The survival rates of mice in sham injury group and hydrogen group during post injury days 7 were 100%. Compared with those in sham injury group, survival rates of mice in pure burn group from post injury days 3 to 7 were significantly decreased (with P values below 0.05). Compared with those in pure burn group, survival rates of mice in burn+ hydrogen group from post injury days 5 to 7 were significantly increased (with P values below 0.05). Conclusions Hydrogen can significantly alleviate the infiltration of inflammatory cells and improve the pathological lesions of lung tissue of mice with severe burn. It has the effects of reducing inflammatory reaction and inhibiting oxidative stress, further showing the protective effect on the lung of burn mice.

Objective To evaluate the effect of hydrogen on autophagy during inflammatory responses following lung injury in burned mice.Methods Ninety-six clean-grade healthy male ICR mice,aged 6 weeks,weighing 20-25 g,were divided into 4 groups (n=24 each) using a random number table:sham operation group (SH group),H2 group (H2 group),burn group (B group) and burn plus H2 group (B+H2 group).Forty percent of the total body surface was shaved with 80 g/L sodium sulfide and then exposed to a 92 ℃ scald device for 18 s in B and B+H2 groups.Forty percent of the total body surface was shaved with 80 g/L sodium sulfide and then exposed to a scald device of skin temperature for 18 s in SH and H2 groups.Mice inhaled 2％ H2 for 1 h starting from 1 and 6 h after burn in H2 and B+H2 groups.The animals were sacrificed at 24 h after burn and lungs were removed for determination of wet/dry weight ratio (W/D rario),expression of autophagy-related microtubule-associated protein 1 light chain 3 (LC3) (by Western blot),activity of myeloperoxidase (MPO),and contents of interleukin-6 (IL-6) and high mobility group box 1 (HMGBI) (by enzyme-linked immunosorbent assay).The ratio of LC3-Ⅱ to LC3-Ⅰ expression (LC3-Ⅱ/LC3-Ⅰ) was calculated.The bronchoalveolar lavage fluid (BALF) was collected at 24 h after burn to detect the concentrations of IL-6 and HMGB1 and to count neutrophil.Results Compared with group SH,the W/D ratio,levels of LC3-Ⅱ/LC3-Ⅰ,MPO,IL-6 and HMGB1,concentrations of IL-6 and HMGB1 in BALF and neutrophil count were significantly increased at 24 h after scald in B and B+H2 groups (P＜0.05).Compared with group B,the W/D ratio,levels of LC3-Ⅱ/LC3-Ⅰ,MPO,IL-6 and HMGB1,concentrations of IL-6 and HMGBl in BALF and neutrophil count were significantly decreased at 24 h after scald in group B+H2 (P＜0.05).Conclusion Hydrogen can alleviate the lung injury in burned mice,and the mechanism is related to enhancing autophagy.

Objective To evaluate the role of nuclear factor erythroid 2-related factor (Nrf2) in hydrogen-induced reduction of intestinal injury in mice with sepsis.Methods Eighteen pathogen-free male wild type mice and 18 male Nrf2 gene knockout mice,aged 6-8 weeks,weighing 20-25 g,were studied.The mice of either type were assigned into 3 groups (n =6 each) using a random number table:sham operation group (group Sham),sepsis group (group Sep) and hydrogen group (group H2).Sepsis was induced by cecal ligation and puncture in chloral hydrate-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 24 h after operation,and small intestinai tissues were removed for determination of the tumor necrosis factor-alpha (TNF-α),interleukin-1beta (IL-1β) and high-mobility group box 1 protein (HMGB 1) contents (by enzyme-linked immunosorbent assay),superoxide dismutase (SOD) and catalase (CAT) activities and malondialdehyde (MDA) content (using a spectrophotometer),8-iso-prostaglandin F2α (8-iso-PGF2α) content (with Multiskan Spectrum plate reader),and expression of heme oxygenase-1 (HO-1) and HMGB1 protein and mRNA (by Western blot or real-time polymerase chain reaction).Results For wild type and Nrf2 gene knockout mice:compared with group Sham,the contents of TNF-α,IL-1β,HMGB1,MDA and 8-iso-PGF2α were significantly increased,the expression of HO-1 and HMGB1 protein and mRNA was up-regulated,and the activities of CAT and SOD were decreased in group Sep (P＜0.05).For wild type mice:compared with group Sep,the levels of TNF-α,IL-1β,MDA and 8-iso-PGF2α were significantly decreased,the expression of HMGB1 protein and mRNA was down-regulated,the expression of HO-1 protein and mRNA was upregulated,and the activities of CAT and SOD were increased in group H2 (P＜0.05).For Nrf2 gene knockout mice:no significant difference was found in the parameters mentioned above between group H2 and group Sep (P＞0.05).Compared with group H2 of wild type mice,the levels of TNF-α,IL-1β,MDA and 8-iso-PGF2α were significantly increased,the expression of HMGB1 protein and mRNA was up-regulated,the expression of HO-1 protein and mRNA was down-regulated,and the activities of CAT and SOD were decreased in group H2 of Nrf2 gene knockout mice (P＜0.05).Conclusion The mechanism by which hydrogen reduces the intestinal injury is related to Nrf2 in mice with sepsis.