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Objective@#To investigate the regulation of hypoxia-inducible factor-1α (HIF-1α) on permeability of rat vascular endothelial cells and the mechanism.@*Methods@#Twelve male Sprague-Dawley rats aged 35 to 38 days were collected and vascular endothelial cells were separated and cultured. The morphology of cells was observed after 4 days of culture, and the following experiments were performed on the 2nd or 3rd passage of cells. (1) Rat vascular endothelial cells were collected and divided into blank control group, negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group according to the random number table (the same grouping method below), with 3 wells in each group. Cells in negative control group, HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were transfected with GV248 empty plasmid, recombinant plasmid respectively containing HIF-1α interference sequence 1, interference sequence 2, and interference sequence 3 with liposome 2000. Cells in blank control group were only transfected with liposome 2000. After transfection of 24 h, expression levels of HIF-1α mRNA and protein of cells in each group were respectively detected by reverse transcription real-time fluorescent quantitative polymerase chain reaction and Western blotting (the same detecting methods below) . The sequence with the highest interference efficiency was selected. (2) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α low expression group, with 3 wells in each group. Cells in blank control group were only transfected with liposome 2000, and cells in negative control group and HIF-1α low expression group were respectively transfected with GV248 empty plasmid and low expression HIF-1α recombinant plasmid selected in experiment (1) with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α in each group were detected. (3) Another batch of rat vascular endothelial cells were collected and divided into blank control group, negative control group, and HIF-1α high expression group, with 3 wells in each group. Cells in blank control group were transfected with liposome 2000, and cells in negative control group and HIF-1α high expression group were respectively transfected with GV230 empty plasmid and HIF-1α high expression recombinant plasmid with liposome 2000. After 14 days of culture, the mRNA and protein expressions of HIF-1α of cells in each group were detected. (4) After transfection of 24 h, cells of three groups in experiment (1) and three groups in experiment (2) were collected, and mRNA and protein expressions of myosin light chain kinase (MLCK), phosphorylated myosin light chain (p-MLC), and zonula occludens 1 (ZO-1) of cells were detected. Data were processed with one-way analysis of variance and t test.@*Results@#After 4 days of culture, the cells were spindle-shaped, and rat vascular endothelial cells were successfully cultured. (1) The interference efficiencies of HIF-1α of cells in HIF-1α interference sequence 1 group, HIF-1α interference sequence 2 group, and HIF-1α interference sequence 3 group were 47.66%, 45.79%, and 62.62%, respectively, and the interference sequence 3 group had the highest interference efficiency. After transfection of 24 h, the mRNA and protein expression levels of HIF-1α of cells in interference sequence 3 group were significantly lower than those in blank control group (t=18.404, 9.140, P<0.01) and negative control group (t=15.099, 7.096, P<0.01). (2) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=21.140, 5.440, P<0.01) and negative control group (t= 14.310, 5.210, P<0.01). (3) After cultured for 14 days, the mRNA and protein expression levels of HIF-1α of cells in HIF-1α high expression group were significantly higher than those in blank control group (t=19.160, 7.710, P<0.01) and negative control group (t= 19.890, 7.500, P<0.01). (4) After transfection of 24 h, the mRNA expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=2.709, 4.011, P<0.05 or P<0.01) and negative control group (t=2.373, 3.744, P<0.05 or P<0.01). The mRNA expression level of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (t=4.285, 5.050, P<0.01). The mRNA expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were significantly higher than those in blank control group (t=9.118, 11.313, P<0.01) and negative control group (t=9.073, 11.280, P<0.01). The mRNA expression level of ZO-1 of cells in HIF-1α high expression group was significantly lower than that in blank control group and negative control group (t=2.889, 2.640, P<0.05). (5) After transfection of 24 h, the protein expression levels of MLCK and p-MLC of cells in HIF-1α low expression group were significantly lower than those in blank control group (t=2.652, 3.983, P<0.05 or P<0.01) and negative control group (t=2.792, 4.065, P<0.05 or P<0.01). The protein expression of ZO-1 of cells in HIF-1α low expression group was significantly higher than that in blank control group and negative control group (t=3.881, 3.570, P<0.01). The protein expression levels of MLCK and p-MLC of cells in HIF-1α high expression group were 1.18±0.24 and 0.68±0.22, which were significantly higher than 0.41±0.21 and 0.35±0.14 in blank control group (t=5.011, 3.982, P<0.05 or P<0.01) and 0.43±0.20 and 0.36±0.12 in negative control group (t= 4.880, 3.862, P<0.05 or P<0.01). The protein expression level of ZO-1 of cells in HIF-1α high expression group was 0.08±0.06, which was significantly lower than 0.20±0.09 in blank control group and 0.19±0.09 in negative control group (t=4.178, 3.830, P<0.05 or P<0.01).@*Conclusions@#HIF-1α up-regulates expressions of MLCK and p-MLC and down-regulates expression of ZO-1, thereby increasing the permeability of rat vascular endothelial cells.
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Objective To explore the mechanism of hypertonic salt solution (HS) alleviates lung injury of rats at the early stage of severe scald.Methods Thirty-two female Sprague-Dawley (SD) rats were randomly assigned to sham group, lactated Ringer solution (LR) group, HS200 group (200 mmol/L HS group, 1 L 200 mmol/L HS contained 955 mL LR and 45 mL 10％ NaCl) and HS400 group (400 mmol/L HS group, 1 L 400 mmol/L HS contained 846 mL LR and 154 mL 10％ NaCl), with 8 rats in each group. A 30％ total body surface area (TBSA)Ⅲ degree scalded model was reproduced by scalded on the back with 98℃ boiling water for 12 seconds, whereas those in the sham group were exposed to 37 ℃ water without liquid resuscitation. Rats in the three drug intervention groups were resuscitated with LR, 200 mmol/L HS and 400 mmol/L HS by caudal vein according to the Parkland formula, respectively. All rats were sacrificed at 8 hours after scald injury to harvest abdominal aorta blood and lung tissues. Interleukins (IL-6, IL-10 and IL-17) in serum were determined by enzyme-linked immunosorbent assay (ELISA). Samples from the lung tissue were used to measure malondialdehyde (MDA) and superoxide dismutase (SOD) levels by ultraviolet spectrophotometer. Expressions of p38 mitogen-activated protein kinase (p38MAPK) and extracellular regulated protein kinase 1/2 (ERK1/2) in the lung were determined by Western Blot. The lung tissue was stained with hematoxylin and eosin (HE), and the pathological changes were observed with a light microscope.Results Compared with the sham group, the lung tissues in the LR group were damage obviously, which accompanied with more inflammatory cell infiltration, cell edema and pulmonary septum thickening, and the levels of IL-6, IL-10, IL-17 in serum and MDA content, the phosphorylation of p38MAPK and ERK1/2 in lung tissues were increased whereas the activity of SOD was decreased. Compared with the LR group, the lung injury was significantly alleviated, the levels of IL-6, IL-17 in serum and MDA content and the phosphorylation of p38MAPK and ERK1/2 were decreased, and the levels of IL-10 and SOD were increased in both HS groups with a dose-dependent manner. There were significant difference in above parameters between HS400 group and LR group [serum IL-6 (ng/L): 3.76±0.12 vs. 6.72±0.90, serum IL-10 (ng/L): 33.76±3.71 vs. 16.77±3.19, serum IL-17 (ng/L): 103.52±2.78 vs. 124.96±4.96, lung MDA (nmol/mg): 5.59±0.24 vs. 7.09±0.39, lung SOD (U/mg):226.7±3.9 vs. 172.7±3.4, lung phosphorylation of p38MAPK (p-p38MAPK)/p38MAPK: 0.15±0.09 vs. 0.35±0.19, lung phosphorylation of ERK1/2 (p-ERK1/2)/ERK1/2: 0.27±0.01 vs. 0.70±0.01, allP < 0.01].Conclusion HS protected against lung injury induced by severe burns in rats with a dose-dependent manner, and it was better than LR, and its possible mechanism was related with reducing the expression of p38MAPK and ERK1/2 pathway in lung tissue, increasing the level of anti-inflammatory cytokines and decreasing the release of pro-inflammatory cytokines, thus inhibiting excessive inflammation and oxidative stress injury in lung.
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Objective To investigate the effect of different concentrations of hypertonic saline solution (HS) on intestine injury in rats at the early stage of severe burn. Methods 104 adult healthy female Sprague-Dawley (SD) rats were randomly divided into five groups: sham group (n = 8), lactated Ringer solution (LR) group (n = 24) and 200, 300, 400 mmol/L HS group (HS200 group, HS300 group, HS400 group, all n = 24). All the rats in LR group and different concentrations of HS groups were scalded for 30% total body surface area (TBSA) with Ⅲ degree, after immediately, the rats were given burn resuscitation therapy by LR or corresponding concentrations of HS through the tail vein. Eight rats were sacrificed on the 2nd, 8th and 24th post-injury hour (PIH), respectively, to collect abdominal aorta blood and intestinal tissues. The rats in sham group were given simulation of burns without resuscitation, which were immediately sacrificed and the specimens were harvested. The serum Na+concentration was determined by automatic biochemical analyzer. Tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) contents in serum were determined by enzyme-linked immunosorbent assay (ELISA). The moisture content of intestine reflected by intestine wet/dry weight (W/D) ratio was determined. The content of malondialdehyde (MDA) and the activity of diamine oxidase (DAO) in intestinal tissue were determined by ultraviolet spectrophotometer. The activation of von Willebrand factor (vWF) was assessed by using immunohistochemistry. Results Compared with sham group, and the contents of TNF-α and IL-1β in blood and W/D ratio and MDA contents in intestine at each time point after injury in LR group and three HS groups were significantly increased, and the activity of intestinal DAO was significantly decreased. The serum Na+concentration was significantly reduced in the LR group as compared with that in the sham group, which was significantly higher in the three HS groups than that in the sham group, with the most obvious change on the 8th PIH. Compared with LR group, the serum Na+concentration and the activity of intestine DAO at each time point after injury in different concentrations of HS groups were significantly increased, and the serum contents of TNF-α, IL-1β and the W/D ratio, MDA contents in intestine were significantly lowered showing a dose dependent. The changes of HS400 group was the most significantly, and the difference on the 8th PIH was statistically significant as compared with LR group [blood Na+(mmol/L): 145.51±0.72 vs. 131.52±0.85, intestinal DAO (U/g): 4.85±0.30 vs. 3.50±0.45, blood TNF-α (ng/L):88.47±4.91 vs. 153.21±13.45, blood IL-1β (ng/L): 85.77±3.42 vs. 140.57±10.46, intestinal W/D ratio: 3.32±0.05 vs. 3.73±0.09, intestinal MDA (nmol/mg): 0.58±0.01 vs. 0.82±0.04, all P < 0.05]. The immunohistochemical results showed that the vWF activity in the LR group and different concentrations of HS groups was significantly reduced as compared with that of the sham group. Compared with LR group, the activity of intestinal vWF at each time point in different concentration of HS groups was increased to some extent with a dose dependent. The positive staining in HS400 group was the deepest, which showed that the activity of intestinal vWF was the strongest after treated by 400 mmol/L HS. Conclusion Compared with LR, HS can attenuate intestinal tissue injury of rats at the early stage of severely burned, and of all, the curative effect of 400 mmol/L HS is the best.
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Objective To investigate the effects of three different concentrations of hypertonic sodium salt (HS) resuscitation on liver injury of rats at the early stage of severe burned.Methods 104 female Sprage-Dawley (SD) rats were randomly divided into five groups: sham group (n = 8), lactated Ringer solution (LR) group (n = 24), 600, 800, 1000 mmol/L HS groups (HS600, HS800, and HS1000 groups,n = 24). Rats in LR group and HS groups were subjected to full-thickness scald with 30% total body surface area (TBSA), and then given liquid resuscitation treatment with LR and the corresponding HS. These rats were sacrificed at 2, 8 and 24 hours post injury to collect blood and liver tissue. Rats in sham group were given simulation of burns without resuscitation, which were immediately sacrificed and the specimens were harvested. The levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected by automatic biochemical analyzer. The levels of liver tissue malondialdehyde (MDA) and superoxide dismutase (SOD) were detected by ultraviolet spectrophotometry. The expression of liver tissue p38 mitogen-actirated protein kinase (p38MARK) was detected by Western Blot.Results Compared with sham group, the levels of ALT, AST, MDA and p38MAPK were increased, and the activities of SOD were decreased in LR group and different degrees in HS groups at each time point after injury. Compared with LR group, the levels of ALT, AST, MDA and p38MAPK were decreased and the activities of SOD were increased in different degrees with HS groups, among which HS600 group changed most significantly [ALT (U/L): 147±52 vs. 227±60 at 8 hours, 138±47 vs. 191±41 at 24 hours; AST (U/L):288±79 vs. 548±237 at 2 hours, 567±167 vs. 841±338 at 8 hours, 515±180 vs. 712±159 at 24 hours; MDA (nmol/mg): 0.287±0.036 vs. 0.395±0.041 at 2 hours, 0.298±0.030 vs. 0.392±0.018 at 8 hours, 0.278±0.033 vs. 0.422±0.036 at 24 hours; SOD (U/mg): 230±16 vs. 159±30 at 2 hours, 251±14 vs. 194±15 at 8 hours, 296±8 vs. 243±11 at 24 hours; p-p38MAPK/p38MAPK (A value): 0.778±0.040 vs. 1.065±0.066 at 2 hours, 0.791±0.046 vs. 0.967±0.041 at 8 hours, 0.733±0.027 vs. 1.020±0.043 at 24 hours; allP < 0.05]. The levels of ALT and AST in HS600 group were significantly lower than those in HS1000 group at 2 hours and in HS800 group at 8 hours. The levels of MDA and p38MAPK in HS600 group were significantly lower than those of HS800 group and HS1000 group, and the level of SOD in HS600 group was significantly higher than that in HS800 group and HS1000 group at each time point after injury. There were no significant differences in all test indicators between HS800 group and HS1000 group at each time point after injury.Conclusions High concentration of HS can reduce the early liver injury in severely scalded rats, of which the curative effect of HS 600 mmol/L is best.
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Objective@#To explore the effects of hypertonic sodium saline (HSS) resuscitation on the liver damage of rats at early stage of severe scald.@*Methods@#Fifty-six SD rats were divided into sham injury group (SI, n=8), lactated Ringer′s solution group (LRS, n=24), and group HSS (n=24) according to the random number table. Rats in group SI were sham injured without resuscitation, while rats in the other two groups were reproduced deep partial-thickness to full-thickness scald model with 30% total body surface area on the back. Rats in group LRS were resuscitated with LRS, while rats in group HSS were resuscitated with 300 mmol/L sodium ion solution according to the Parkland formula. Blood of abdominal aorta and liver of 8 rats in group SI immediately post injury and in the other two groups at post injury hour (PIH) 2, 8, and 24 respectively were collected. Then liver water content was determined by dry-wet weight method. Serum content of alanine aminotransferase (ALT) and aspartate transaminase (AST) was detected by automatic biochemical analyzer. Serum content of tumor necrosis factor α (TNF-α), interleukin-1 (IL-1), and high mobility group box 1 (HMGB1) was determined by enzyme-linked immunosorbent assay. Liver content of malondialdehyde (MDA) and superoxide dismutase (SOD) was detected by ultraviolet spectrophotometer. Pathologic changes of liver were observed by HE staining. Data were processed with one-way analysis of variance and SNK test.@*Results@#(1) At PIH 2, 8, and 24, liver water content of rats in group LRS was higher than that in group SI and group HSS (P<0.05 or P<0.01). (2) At PIH 2, serum ALT content of rats in the three groups was similar (with P values above 0.05). At PIH 8 and 24, serum ALT content of rats in group HSS and group LRS was higher than that in group SI (P<0.05 or P<0.01), and serum ALT content of rats in group HSS was lower than that in group LRS (with P values below 0.01). At PIH 2, 8, and 24, serum AST content of rats in group HSS and group LRS was higher than that in group SI (with P values below 0.01). At PIH 2 and 8, serum AST content of rats in group HSS was lower than that in group LRS (P<0.05 or P<0.01). (3) At PIH 2 and 8, serum TNF-α content of rats in group LRS was (123±39) and (153±38) pg/mL respectively, higher than that in group SI [(60±18) pg/mL] and group HSS [(85±10) and (94±16) pg/mL respectively, with P values below 0.01]. At PIH 8, serum TNF-α content of rats in group HSS was higher than that in group SI (P<0.05). At PIH 24, serum TNF-α content of rats in the three groups was similar (with P values above 0.05). At PIH 2, 8, and 24, serum IL-1 content of rats in group LRS was (122±35), (141±30), and (122±31) pg/mL respectively, and that in group HSS was (80±12), (93±15), and (80±11) pg/mL respectively, all higher than that in group SI [(40±17) pg/mL, with P values below 0.01]; serum IL-1 content of rats in group HSS was lower than that in group LRS (with P values below 0.01). At PIH 2, serum HMGB1 content of rats in the three groups was similar (with P values above 0.05). At PIH 8 and 24, serum HMGB1 content of rats in group LRS was (0.386±0.146) and (0.590±0.188) ng/mL respectively, higher than that in group SI [(0.050±0.027) ng/mL] and group HSS [(0.143±0.038) and (0.309±0.095) ng/mL respectively, with P values below 0.01]. At PIH 24, serum HMGB1 content of rats in group HSS was higher than that in group SI (P<0.01). (4) At PIH 2, 8, and 24, liver MDA content of rats in group HSS and group LRS was higher than that in group SI and their liver SOD content was lower than that in group SI (with P values below 0.01); liver MDA content of rats in group HSS was lower than that in group LRS and their liver SOD content was higher than that in group LRS (with P values below 0.01). (5) Compared with those of rats in group SI, liver cells of rats in group LRS showed massive steatosis at each time point, and liver cell-edema appeared at PIH 8 and 24; while liver cells of rats in group HSS showed little steatosis only at PIH 8 and 24, and the liver cell-edema never appeared.@*Conclusions@#Compared with LRS, HSS resuscitation can alleviate liver injury of rats at the early stage of severe scald through relieving inflammatory mediators and reducing degree of oxidative stress, etc.
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AIM:To study the diagnosis of hyperchloremic triple acid-base disorders (hyperchloremic TABD) and its mechanisms and to find out the cause of hyperchloremic TABD of the burn patients. METHODS:① 154 concomitant arterial blood gas and electrolyte panel of 113 burn patients were diagnosed by clinical approach. ② The plasma aldosterone concentrations of burn patients were tested by radioimmunoassay. RESULTS:9 cases of the 113 burn patients were hyperchloremic TABD. Their plasma aldosterone's values were significantly higher than that of the natural ( P