Effect of calmodulin and its mutants on binding to Na1.2 IQ / 浙江大学学报·医学版

OBJECTIVE@#To investigate the effect of calmodulin (CaM) and its mutants on binding to voltage-gated Na channel isoleucine-glutamine domain (Na1.2 IQ).@*METHODS@#The cDNA of Na1.2 IQ was constructed by PCR technique, CaM mutants CaM, CaM and CaM were constructed with Quickchange site-directed mutagenesis kit (QIAGEN). The binding of Na1.2 IQ to CaM and CaM mutants under calcium and calcium free conditions were detected by pull-down assay.@*RESULTS@#Na1.2 IQ and CaM were bound to each other at different calcium concentrations, while GST alone did not bind to CaM. The binding affinity of CaM and Na1.2 IQ at [Ca]-free was greater than that at 100 nmol/L [Ca] ( < 0.05). In the absence of calcium, the binding amount of CaM wild-type to Na1.2 IQ was greater than that of its mutant, and the binding affinity of CaM to Na1.2 IQ was the weakest among the three mutants ( < 0.05).@*CONCLUSIONS@#The binding ability of CaM and CaM mutants to Na1.2 IQ is Ca-dependent. This study has revealed a new mechanism of Na1.2 regulated by CaM, which would be useful for the study of ion channel related diseases.

Regulation of hypoxia inducible factor-1α on permeability of vascular endothelial cells and the mechanism / 中华烧伤杂志

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.

Analysis of reasons for failure of Meek micro-skin grafting in children with severe burn and treatment measures / 中华烧伤杂志

Objective@#To analyze the reasons for failure of Meek micro-skin grafting in children with severe burns and to observe the clinical effects of the treatment measures.@*Methods@#Thirty children with severe burns hospitalized in the First Affiliated Hospital of Anhui Medical University (hereinafter referred to as the author′s affiliation) from January 2012 to January 2018, conforming to the inclusion criteria were included to failed skin graft group. Children in failed skin graft group were performed with Meek micro-skin grafting operation and the operation failed, including 17 males and 13 females aged 1 to 12 year(s). Thirty children with severe burns hospitalized in the author′s affiliation during the same period of time, conforming to the inclusion criteria, were included to successful skin graft group. Children in successful skin graft group were performed with Meek micro-skin grafting operation and the operation succeeded, including 16 males and 14 females aged 1 to 12 year(s). Main treatment measures and effects before operation, area and survival rate of Meek micro-skin graft, infected pathogens status, selection status of sensitive antibiotics, preoperative nutrition status, and wound infection status in plum rain season of children in the two groups, and nutritional status before and after strengthening nutritional support of postoperative surviving children in failed skin graft group were analyzed retrospectively. Data were processed with chi-square test and t test.@*Results@#(1) The numbers of children in the two groups performed with main treatment measures of dilatation and anti-shock, tracheotomy intubation, ventilator-assisted respiration, and limb incision decompression after admission were close (χ2=0, 0.016, 0.025, 0.009, P>0.05). After taking the above-mentioned main treatment measures, effects of correcting shock, preventing asphyxia, correcting breathing difficulty, and improving peripheral circulation of limb were achieved. (2) The area of Meek micro-skin grafting of children in successful skin graft group was (20.6±2.5)% total body surface area (TBSA), close to (21.2±2.2)% TBSA in failed skin graft group (t=0.534, P>0.05). The survival rate of Meek micro-skin graft of children in successful skin graft group was (79±5)%, significantly higher than (26±3)% in failed skin graft group (t=2.956, P<0.01). (3) The microbial culture of wound secretion of 5 (16.67%) children in 30 patients in successful skin graft group was positive, with Pseudomonas aeruginosa of 2 children, and Escherichia coli, Staphylococcus aureus, and Aspergillus of one patient respectively. As children in successful skin graft group were with no symptom of systemic infection, no blood microbial culture was done. The microbial culture of wound secretion of 30 (100.00%) children in 30 patients in failed skin graft group was positive, and blood microbial culture of 8 (26.67%) children was positive. The main pathogen was Pseudomonas aeruginosa of 11 (36.67%) children in 8 pathogens caused infection with gram-negative bacteria of 22 (73.33%), gram-positive bacteria of 11 (36.67%) children, and fungi of 6 (20.00%) children. (4) Ten kinds of sensitive antibiotics such as cephalosporins, glycopeptides, carbapenems, and tetracyclines antibiotics were used in children in failed skin graft group, of which the use rate of imipenem of 9 (30.00%) was the highest. Only 4 kinds of sensitive antibiotics such as ceftazidime were used in 30 children in successful skin graft group. (5) The preoperative levels of albumin and prealbumin of children in successful skin graft group were (32±4) g/L and (133±41) mg/L respectively, significantly higher than (27±4) g/L and (93±35) mg/L in failed skin graft group (t=5.090, 4.064, P<0.01). The albumin and prealbumin levels of postoperative surviving children in failed skin graft group after nutritional support treatment were (35±4) g/L and (168±49) mg/L, significantly higher than (27±4) g/L and (94±38) mg/L before nutritional support treatment (t=6.911, 6.315, P<0.01). (6) Wound infection of 9 children in 30 children with wound infection in failed skin graft group happened in the plum rain season, and fungi infection of 3 children in 6 children with fungi infection happened in the plum rain season. Wound infection of 2 children in 5 children with wound infection in successful skin graft group happened in the plum rain season, and the only one children with fungi infection happened in the plum rain season.@*Conclusions@#The main reasons for the failure of Meek micro-skin grafting in children with severe burns include infection, nutrition, and season factors, etc. Measures of strengthening wound dressing change, reasonable use of sensitive antibiotics to control infection, internal and external intestinal nutritional support, and reducing disturbance of the plum rain season by enhancing ventilation are effective and worthy of clinical promotion.

Study on the mechanism of hypertonic salt solution alleviates lung injury of rats at the early stage of severe scald / 中华危重病急救医学

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.

Influence of three-level collaboration network of pediatric burns treatment in Anhui province on treatment effects of burn children / 中华烧伤杂志

Objective@#To explore the influence of three-level collaboration network of pediatric burns in Anhui province on treatment effects of burn children.@*Methods@#The data of medical records of pediatric burn children transferred from Lu′an People′s Hospital and Fuyang People′s Hospital to the First Affiliated Hospital of Anhui Medical University from January 2014 to December 2015 and January 2016 to September 2017 (before and after establishing three-level collaboration network of pediatric burns treatment) were analyzed: percentage of transferred burn children to hospitalized burn children in corresponding period, gender, age, burn degree, treatment method, treatment result, occurrence and treatment result of shock, and operative and non-operative treatment time and cost. Rehabilitation result of burn children transferred back to local hospitals in 2016 and 2017. Data were processed with t test, chi-square test, Mann-Whitney U test, and Fisher′s exact test.@*Results@#(1) Percentage of burn children transferred from January 2014 to December 2015 was 34.3% (291/848) of the total number of hospitalized burn children in the same period of time, which was close to 30.4% (210/691) of burn children transferred from January 2016 to September 2017 (χ2=2.672, P>0.05). (2) Gender, age, burn degree, and treatment method of burn children transferred from the two periods of time were close (χ2=3.382, Z=-1.917, -1.911, χ2=3.133, P>0.05). (3) Cure rates of children with mild, moderate, and severe burns transferred from January 2016 to September 2017 were significantly higher than those of burn children transferred from January 2014 to December 2015 (χ2=11.777, 6.948, 4.310, P<0.05). Cure rates of children with extremely severe burns transferred from the two periods of time were close (χ2=1.181, P>0.05). (4) Children with mild and moderate burns transferred from the two periods of time were with no shock. The incidence of shock of children with severe burns transferred from January 2014 to December 2015 was 6.0% (4/67), and 3 children among them were cured. The incidence of shock of children with severe burns transferred from January 2016 to September 2017 was 3.9% (2/51), and both children were cured. The incidences and cures of shock of children with severe burns transferred from the two periods of time were close (χ2=0.006, P>0.05). Incidence of shock of children with extremely severe burns transferred from January 2014 to December 2015 was 57.1% (32/56), significantly higher than that of burn children transferred from January 2016 to September 2017 [34.5% (10/29), χ2=3.925, P<0.05]. Shock of 25 children with extremely severe burns transferred from January 2014 to December 2015 were cured, and shock of 9 children with extremely severe burns transferred from January 2016 to September 2017 were cured. The cures of shock of children with extremely severe burns transferred from the two periods of time were close ( χ2=0.139, P>0.05). (5) Time of operative treatment of children with moderate, severe, and extremely severe burns transferred from January 2014 to December 2015 was obviously longer than that of burn children transferred from January 2016 to September 2017 (t=2.335, 2.065, 2.310, P<0.05). Time of operative treatment of children with mild burns transferred from the two periods of time was close (Z=-0.417, P>0.05). Costs of operative treatment of children with moderate and severe burns transferred from January 2014 to December 2015 were significantly more than those of burn children transferred from January 2016 to September 2017 (Z=-3.324, t=2.167, P<0.05). Costs of operative treatment of children with mild and extremely severe burns transferred from the two periods of time were close (t=0.627, 0.808, P>0.05). (6)Time of non-operative treatment of children with mild, moderate, and severe burns transferred from January 2014 to December 2015 was obviously longer than that of burn children transferred from January 2016 to September 2017 (t=2.335, Z=-2.095, t=2.152, P<0.05). Time of non-operative treatment of children with extremely severe burns transferred from the two periods of time was close (t=0.450, P>0.05). Costs of non-operative treatment of children with moderate and severe burns transferred from January 2014 to December 2015 were obviously higher than those of burn children transferred from January 2016 to September 2017 (Z=-2.164, t=2.040, P<0.05). Costs of non-operative treatment of children with mild and extremely severe burns transferred from the two periods of time were close (t=0.146, 1.235, P>0.05). (7) Sixty-seven burn children transferred from January 2016 to September 2017 were transferred back to local hospitals for rehabilitation under the guidance of experts of the First Affiliated Hospital of Anhui Medical University, with 25 patients in 2016 and 42 patients in 2017. Effective rehabilitation rates of burn children transferred back to local hospitals for rehabilitation in 2016 and 2017 were both 100%.@*Conclusions@#The three-level collaboration network of pediatric burns treatment in Anhui province can effectively increase cure rate of children with mild, moderate, and severe burns, reduce incidence of shock of children with extremely severe burns, shorten time of operative treatment of burn children with moderate, severe, and extremely severe burns, and time of non-operative treatment of children with mild, moderate, and severe burns, reduce treatment costs of children with moderate and severe burns, and improve rehabilitation effectiveness of children transferred from Lu′an People′s Hospital and Fuyang People′s Hospital to the the First Affiliated Hospital of Anhui Medical University.

Clinical analysis of the synthesized treatment for elderly patients with serious burning / 中华老年医学杂志

Objective To summarize the experience of early management in elderly patients with serious burning in order to raise cure rate. Methods Elderly patients (63 cases) with serious burning (burned area exceeding 30% or Ⅲ? area exceeding 10% ) admitted from 1990 to 2002 were retrospectively studied. Patients were divided into two groups according to the admission date, before and after January 1, 1996 since new management was extensively applied after 1996. These measures consisted of rapid and adequate fluid resuscitation, early enteral feeding and autograft after eschar excision. Results The incidence of sepsis and MODS was 38.7% and 19.4% respectively after 1996. It was evidently lower than the group before 1996(65.6% vs 43.8%, P