中华烧伤与创面修复杂志

2021, 37(9): F01-F01. doi: 10.3760/cma.j.issn.1009-2587.2021.09.101

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Metabolic staging and nutritional treatment strategies of severely burned patients

Peng Xi

2021, 37(9): 805-810. doi: 10.3760/cma.j.cn501120-20210802-00264

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The complexity of burn metabolism and limitations of its cognition are the core factors leading to dissatisfactory efficacy of nutritional therapy in severe burn patients. Precise understanding of burn metabolic rules is a prerequisite for improving the pertinence and effectiveness of nutritional therapy. The previous division of burn metabolic stages based on energy consumption did not pay enough attention to substance metabolism, so it is difficult to fully comprehend the overall change pattern and stage characteristics of burn metabolism. Through the effective integration of the metabonomics, physiomics, and clinical data, combining the pathophysiological characteristics of burns at different clinical phases, this paper suggested that the metabolism of severe burns should be divided into four stages and proposed the corresponding nutritional strategies and measures according to different metabolic characteristics, in order to provide a basis for further enhancing the nutritional efficacy in burn patients.

Study on the effect and mechanism of recombinant human intestinal trefoil factor on intestinal mucosal injury and repair in burned mice

Wu Dan, Wang Chao, Li Teng, Wang Huan, Hu Jianhong, Peng Xi

2021, 37(9): 811-820. doi: 10.3760/cma.j.cn501120-20210412-00125

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Objective To establish an efficient human intestinal trefoil factor (ITF) recombinant expression and purification strategy and to observe the effect of recombinant human ITF (rhITF) on intestinal mucosal injury and repair in burned rats and to explore the mechanism. Methods The experimental research method was applied. New yeast expression vector pGAPZαA and yeast X33 were used to express recombinant ITF. The protein was purified by metal chelation affinity chromatography and anion and cation exchange chromatography. The rhITF was identified by non-reductive sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western-blotting. The rhITF was mixed with pepsin solution and trypsin solution in a volume ratio of 1∶1, respectively. After mixed with pepsin solution for 0.5, 1.0, 1.5, 2.0 h and trypsin solution for 1.0, 2.0, 4.0 h, the stability of rhITF was analyzed with non-reductive SDS-PAGE. One hundred and five male BALB/c mice aged 6-8 weeks were divided into sham injury group (n=30), burn alone group (n=45), and burn+rhITF group (n=30) according to the random number table. Mice in burn alone group and burn+rhITF group were inflicted with 30% total body surface area full-thickness burn on the back, while mice in sham injury group were simulated with burn. After burn, mice in burn+rhITF group were intragastrically administered with rhITF of 1 mg/kg, while mice in the other two groups were given the same amount of normal saline. At post injury hour 24, 15 mice in burn alone group were collected to prepare burn serum, which was used in the cell experiment. On post injury day (PID) 3, 5, and 7, 10 mice in each group were sacrificed to collect the small intestinal tissue. The pathological changes of the intestinal mucosa were observed by hematoxylin-eosin staining, and the activities of diamine oxidase (DAO) and lactic dehydrogenase (LDH) in the intestinal tissue were determined by spectrophotometry and enzyme linked immunosorbent assay. Three batches of human colorectal adenocarcinoma HT-29 cells were taken and divided into negative control group, 25 μg/mL rhITF group, 50 μg/mL rhITF group (n=3), normal control group, burn serum group, burn serum+rhITF group (n=3), and CK869 inhibitor group, CK666 inhibitor group, solvent control group (n=2), respectively, which were dealt with the corresponding treatment. After 12 h of culture, the migration of cells were observed by Transwell experiment. Another 2 batches of HT-29 cells were taken and each batch of cells were divided into normal control group, burn serum group, and burn serum+rhITF group (n=6). After 24 h of culture, the protein expressions of adenosine monophosphate activated protein kinase (AMPK), phosphorylated AMPK (p-AMPK), Ras related C3 botulinum toxin substrate 1 (Rac1), and actin-related protein 2/3 (Arp 2/3) complex, subunit 1B (ARPC1B) in the cells were detected by Western blotting, and the Rac1 activity of the cells was detected by activated magnetic bead pull-down test. Data were statistically analyzed with analysis of variance for factorial design, one-way analysis of variance, and Student-Newman-Keuls test. Results Totally 82.35 mg rhITF was gathered from per litre of fermentation broth with protein purity up to 98%, and the rhITF had good antigenicity. The rhITF was stable in pepsin solution and trypsin solution, with 45% rhITF remained after 2.0 h in trypsin solution, and there was 90% rhITF remained after 4.0 h in pepsin solution. At each time point post injury, no hyperemia, or edema was observed in intestinal mucosa of mice in sham injury group, the main pathological manifestations of intestinal mucosa in mice of burn alone group were hyperemia, edema, erosion, and hemorrhage, and the main manifestations of intestinal mucosa of mice in burn+rhITF group were hyperemia and edema on PID 3 and 5, which were alleviated on PID 7. Compared with those of burn alone group, the activities of DAO and LDH in intestinal tissue of mice in sham injury group and burn+rhITF group were significantly increased on PID 3, 5, and 7 (P<0.05 or P<0.01 ). After 12 h of culture, the number of cell migration in 25 μg/mL rhITF group was 58±12, which was obviously more than 16±5 in negative control group (P<0.01) and obviously less than 123±9 in 50 μg/mL rhITF group (P<0.05). After 12 h of culture, the number of cell migration in burn serum group was 60±13, which was significantly less than 143±11 in normal control group and 138±8 in burn serum+rhITF group (P<0.05). After 12 h of culture, the number of cell migration in solvent control group was 155±9, which was significantly more than 33±5 in CK666 inhibitor group and 28±5 in CK869 inhibitor group (P<0.01). After 24 h of culture, the protein expressions of AMPK and Rac1 of cells in burn serum group were close to those of normal control group and burn serum+rhITF group (P˃0.05), the protein expression of p-AMPK of cells in burn serum group was significantly higher than that of normal control group and burn serum+rhITF group, respectively (P<0.05 or P<0.01), and the protein expression of ARPC1B of cells in burn serum group was significantly lower than that of normal control group and burn serum+rhITF group (P<0.05). After 24 h of culture, the Rac1 activity of cells in burn serum group was significantly lower than that in normal control group and burn serum+rhITF group, respectively (P<0.05 or P<0.01). Conclusions The rhITF obtained in this study has high purity and super stability, which can resist extreme pH and hydrolysis of protease and can relieve intestinal mucosal damage in burned mice. The rhITF can promote the migration of intestinal epithelial cells and accelerate the repair of intestinal mucosa through inhibiting phosphorylation of AMPK to maintain Rac1-Arp2/3 activity.

A prospectively randomized controlled study of the effects of intensive insulin therapy combined with glutamine on nutritional metabolism, inflammatory response, and hemodynamics in severe burn patients

Lou Jiaqi, Li Ye, Cui Qingwei, Zhang Pan, Sun Han, Tang Hao, Zhuang Mengmeng, Sun Yong

2021, 37(9): 821-830. doi: 10.3760/cma.j.cn501120-20210428-00159

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Objective To observe the effects of intensive insulin therapy combined with glutamine on nutritional metabolism, inflammatory response, and hemodynamics in severe burn patients. Methods Thirty-two severe burn patients who met the inclusion criteria and hospitalized in the Affiliated Huaihai Hospital of Xuzhou Medical University from June 2017 to January 2019 were recruited into a prospectively randomized controlled study. According to the random number table, the patients were divided into conventional insulin therapy alone group, conventional insulin therapy+glutamine group, intensive insulin therapy alone group, and intensive insulin therapy+glutamine group, with 8 patients in each group, with genders of 5 males and 3 females, 4 males and 4 females, 3 males and 5 females, 4 males and 4 females, and ages of (35±7), (36±9), (33±11), and (38±7) years, respectively. Patients in conventional insulin therapy alone group were treated with conventional insulin therapy on the basis of routine treatment to control the blood glucose. Patients in conventional insulin therapy+glutamine group were supplemented with alanyl-glutamine for more than 14 days in addition to the treatment in conventional insulin therapy alone group. Patients in intensive insulin therapy alone group were treated with intensive insulin therapy on the basis of routine treatment to control the blood glucose. Patients in intensive insulin therapy+glutamine group were supplemented with alanyl-glutamine in addition to the treatment in intensive insulin therapy alone group. On treatment day (TD) 1, 3, 7, and 14, the blood glucose, albumin, prealbumin, white blood cell count, procalcitonin (PCT), and C-reactive protein (CRP) of patients in the 4 groups were detected. The cardiac index (CI), stroke volume index (SVI), global end-diastolic volume index (GEDVI), systemic vascular resistance index (SVRI), extravascular lung water index (EVLWI), and pulmonary vascular permeability index (PVPI) of patients in the 4 groups on TD 1, 3, and 7 were measured. Data were statistically analyzed with Fisher′s exact probability test, one-way analysis of variance, analysis of variance for repeated measurement, and Bonferroni method. Results All patients in the 4 groups successfully completed the study, and there were no withdrawal cases. On TD 3, 7, and 14, the blood glucose of patients in intensive insulin therapy alone group ((5.9±1.3), (5.8±0.6), (5.5±0.5) mmol/L) and intensive insulin therapy+glutamine group ((5.9±1.1), (5.6±1.1), (5.2±0.8) mmol/L) were significantly lower than those in conventional insulin therapy alone group ((9.1±0.5), (8.4±0.9), (7.4±1.1) mmol/L, P<0.05). Compared with those in conventional insulin therapy alone group, the levels of albumin of patients in conventional insulin therapy+glutamine group, intensive insulin therapy alone group, and intensive insulin therapy+glutamine group were significantly increased on TD 7 and 14 (P<0.05). Compared with the level of albumin of patients in intensive insulin therapy+glutamine group, the levels of albumin of patients in conventional insulin therapy+glutamine group and intensive insulin therapy alone group were significantly decreased on TD 14 (P<0.05). Compared with those in conventional insulin therapy alone group, the levels of prealbumin of patients in conventional insulin therapy+glutamine group and intensive insulin therapy alone group were significantly increased on TD 7 and 14 (P<0.05). Compared with those in intensive insulin therapy+glutamine group, the levels of prealbumin of patients in intensive insulin therapy alone group and conventional insulin therapy+glutamine group were significantly decreased on TD 1, 7, and 14 (P<0.05). There were no statistically significant differences in the white blood cell count, PCT, and CRP of patients in the 4 groups in pairwise comparison between groups on TD 1, 3, 7, and 14 (P>0.05). On TD 3 and 7, the levels of cardiac index, SVI, GEDVI, and SVRI of patients in intensive insulin therapy+glutamine group were significantly higher than those in conventional insulin therapy alone group (P<0.05), while the levels of EVLWI and PVPI were significantly lower than those in conventional insulin therapy alone group (P<0.05). Conclusions Glutamine combined with intensive insulin therapy can improve the hypermetabolism in patients after severe burns, reduce the decomposition and consumption of endogenous nutrient substrates, and at the same time help the recovery of cardiac function and maintenance of hemodynamic stability.

Analysis of risk factors of early enteral nutrition intolerance in extremely severe burn patients

Pan Yanyan, Xu Sida, Fan Youfen, Tu Jing, Huang Neng, Yu Yaohua, Cui Shengyong, Le Xin, Xu Pei, Jin Guoying, Chen Cui

2021, 37(9): 831-838. doi: 10.3760/cma.j.cn501120-20210511-00180

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Objective To explore the risk factors of early enteral nutrition intolerance in extremely severe burn patients. Methods A retrospective case-control study was performed. From January 2018 to December 2020, seventy-six adult patients with extremely severe burns who met the inclusion criteria were admitted to Hwa Mei Hospital of University of Chinese Academy of Sciences, including 55 males and 21 females, aged (45±11) years with burns of 62% (52%, 82%) total body surface area. Depending on the patient's tolerance to early enteral nutrition, they were divided into tolerance group (47 patients) and intolerance group (29 patients), and their clinical data were statistically analyzed, including age, sex, body mass index (BMI), underlying disease, total burn area, full-thickness burn area, abbreviated burn severity index (ABSI) score, implementation of mechanical ventilation on the day of admission, stable shock state, vomiting before feeding. The following data were recorded including the onset time, duration length, and frequency of enteral nutrition intolerance of patients in intolerance group, and the number of operations, the length of hospitalization, the occurrence of sepsis within 2 weeks after injury, the outcome, as well as the serum hypersensitive C-reactive protein (hs-CRP), albumin, fasting blood glucose, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and γ-glutamyl transpeptidase (γ-GT) on post burn day (PBD) 1, 5, 9, and 13 of patients in the two groups. Data were statistically analyzed with independent sample t test, Mann-Whitney U test, and chi-square test to screen the related factors of early enteral nutrition intolerance of the patients. Binary univariate and multivariate logistic regression analysis were used to analyze the independent risk factors of early enteral nutrition intolerance of the patients. Results There were no statistically significant differences in age, sex, BMI, and percentage of underlying disease of patients between the two groups (P>0.05). The total burn area, full-thickness burn area, ABSI score, mechanical ventilation percentage on the day of admission, percentage of unstable shock period, percentage of vomiting before feeding of patients in intolerance group were significantly higher than those in tolerance group (Z=-4.559, -3.378, -4.067, χ²=18.375, 23.319, 8.339, P<0.01). In intolerance group, the onset time of intolerance was (9±4) d after injury, and the duration length was 4 (2, 6) d, with a total of 46 times occurred. Compared with tolerance group, the percentage of sepsis and mortality of patients in intolerance group were significantly higher within 2 weeks after injury (χ²=16.571, 12.665, P<0.01). The number of operation and length of hospitalization of patients in the two groups were similar (P>0.05); however the length of hospitalization of patients in the intolerance group was significantly more than that in tolerance group after excluding the death cases (Z=-2.266, P<0.05). On PBD 1, the level of fasting blood glucose and AST of patients in intolerance group were significantly higher than those in tolerance group (t=3.070, Z=-3.070, P<0.01). On PBD 5, the levels of hs-CRP, albumin, fasting blood-glucose, ALT, AST, and γ-GT of patients in the two groups were similar (P>0.05). On PBD 9, the level of hs-CRP of patients in intolerance group was significantly higher than that in tolerance group (t=2.836, P<0.01), and the levels of ALT and γ-GT of patients in intolerance group were significantly lower than those in tolerance group (Z=-3.932, -2.052, P<0.05 or P<0.01). On PBD 13, the level of hs-CRP of patients in intolerance group was significantly higher than that in tolerance group (t=3.794, P<0.01), and the levels of fasting blood glucose, ALT, and γ-GT of patients in intolerance group were significantly lower than those in tolerance group (t=-2.176, Z=-2.945, -2.250, P<0.05 or P<0.01). Binary univariate logistic regression analysis showed that total burn area, full-thickness burn area, ABSI score, implementation of mechanical ventilation on the day of admission, unstable shock period, vomiting before feeding, and fasting blood-glucose on PBD 1 of patients were related to early enteral nutrition intolerance (odds ratio=1.086, 1.052, 1.775, 9.167, 12.797, 10.125, 1.249, 95% confidence interval=1.045 - 1.129, 1.019 - 1.085, 1.320 - 2.387, 3.132 - 26.829, 4.199 - 39.000, 2.003 - 51.172, 1.066 - 1.464, P<0.01). Multivariate logistic regression analysis showed that the large total burn area, unstable shock period, vomiting before feeding, and high fasting blood-glucose on PBD 1 of patients were the independent risk factors of early enteral nutrition intolerance in patients (odds ratio=1.073, 6.390, 9.004, 1.246, 95% confidence interval=1.021 - 1.128, 1.527 - 26.734, 1.134 - 71.496, 1.007 - 1.540, P<0.05 or P<0.01). Conclusions The percentage of early enteral nutrition intolerance is very high in extremely severe burn patients, which is closely related to poor prognosis. Large total burn area, vomiting before feeding, unstable shock phase, high fasting glucose on PBD 1 of patients are the independent risk factors for early enteral nutrition intolerance in extremely severe burn patients. The benefits and risks should be carefully evaluated before starting enteral nutrition in such patients, and early enteral nutrition should not be blindly pursued.

Changes in the related indicators of bone formation and bone resorption in severely burned rats

Gong Xiang, Ye Ziqing, Yu Gang, Zhang Wei, Zhang Weidong, Zhou Xueqing, Li Min, Xie Weiguo

2021, 37(9): 839-845. doi: 10.3760/cma.j.cn501120-20200505-00253

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Objective To observe the changes in the related indicators of bone formation and bone resorption in severely burned rats. Methods The experimental research method was adopted. Thirty female Sprague-Dawley rats aged 6 to 8 weeks were divided into sham injury group, 12% total body surface area (TBSA) full-thickness burn group, and 24%TBSA full-thickness burn group according to the random number table, with 10 rats in each group. The rats were treated on the back correspondingly, after which, the burned rats were rehydrated by intraperitoneal injection according to the Parkland formula, and the wound was coated with 20 g/L iodophor until wound healing. On post injury day (PID) 28, the tibia tissue of rats in each group was collected. The new bone tissue and the number of osteoclasts were observed after staining with Masson and tartrate-resistant acid phosphatase, respectively. The abdominal aortic blood of rats in each group was harvested for serum preparation. The bone metabolism indexes of serum calcium ion and phosphorus ion concentration were determined by the methyl thymol blue colorimetric method and phosphomolybdic acid method, respectively. The serum levels of bone formation marker of aminoterminal propeptide of type 1 procollagen (P1NP) and bone resorption marker of beta-carboxy-terminated peptide of type Ⅰ collagen (β-CTX) were determined by enzyme-linked immunosorbent assay. The first lumbar spine tissue of rats in each group was collected, and the mRNA expression levels of osteoprotegerin, receptor activator of nuclear factor-κB ligand (RANKL), tumor necrosis factor receptor-associated factor 6 (TRAF-6), nuclear factor of activated T cell 1 (NFATC1), c-Fos, and c-Src were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were statistically analyzed with one-way analysis of variance, Bonferroni method, Welch test, Games-Howell test, Kruskal-Wallis test, Mann-Whitney U test, and Bonferroni correction. Results On PID 28, compared with that in sham injury group, the formation of new bone tissue in the tibia tissue of rats in the two burn groups was decreased, and the larger the burn area, the more obvious the decrease. The numbers of osteoclasts in the tibia tissue of rats in the two burn groups were similar, both significantly more than the number in sham injury group. On PID 28, the serum calcium ion concentration and serum level of β-CTX of rats in the three groups were similar (P>0.05). The serum phosphorus ion concentration of rats in 24%TBSA full-thickness burn group was significantly higher than that in 12%TBSA full-thickness burn group (P<0.05), and the serum phosphorus ion concentrations in the two burn groups were significantly higher than the concentration in sham injury group (P<0.01). The serum level of P1NP of rats in 24%TBSA full-thickness burn group was significantly lower than that in sham injury group (P<0.01). On PID 28, the mRNA expression levels of osteoprotegerin in the first lumbar spine tissue of rats in sham injury group, 12%TBSA full-thickness burn group, and 24%TBSA full-thickness burn group were 1.01±0.20, 1.71±0.83, and 2.24±0.51, respectively, and that in 24%TBSA full-thickness burn group was significantly higher than that in sham injury group (P<0.01). The mRNA expression level of RANKL in the first lumbar spine tissue of rats in 24%TBSA full-thickness burn group was 1.31±0.17, which was significantly higher than 1.00±0.14 in sham injury group and 0.97±0.10 in 12%TBSA full-thickness burn group (P<0.01). The mRNA expression levels of TRAF-6, NFATC1 (Z=3.141, 3.782), and c-Src in the first lumbar tissue of rats in 12%TBSA full-thickness burn group and 24%TBSA full-thickness burn group and the mRNA expression level of c-Fos in the first lumbar tissue of rats in 12%TBSA full-thickness burn group were significantly higher than those in sham injury group (P<0.05 or P<0.01). The mRNA expression levels of c-Fos and c-Src in the first lumbar spine tissue of rats in 12%TBSA full-thickness burn group were significantly higher than those in 24%TBSA full-thickness burn group (P<0.01). Conclusions Severe burns can cause a decrease in the generation of new bone tissue, an increase in the number of osteoclasts and the serum phosphorus ion concentration, and a decrease in the serum level of P1NP in rats. The level of osteoprotegerin, RANKL, TRAF-6, NFATC1, c-Fos, and c-Src in bone tissue showed an increasing trend while the level of NFATC1, c-Fos, and c-Src showed a decreasing trend with the increase of burn area.

Establishment and validation of a clinical prediction model for infection risk at the placement sites of skin and soft tissue expanders

Dong Chen, Yu Zhou, Liu Wei, Liu Hengxin, Tang Yinke, Ma Xianjie

2021, 37(9): 846-852. doi: 10.3760/cma.j.cn501120-20200619-00314

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Objective To establish a clinical prediction model for infection risk at the placement sites of skin and soft tissue expanders (hereinafter termed as expanders) and to validate the predictive value of the model. Methods A retrospective observational study was conducted. Totally 2 934 patients who underwent skin and soft tissue dilatation surgery in the Department of Plastic Surgery of the First Affiliated Hospital of Air Force Medical University from January 2009 to December 2018 and met the selection criteria were included. There were 1 867 males and 1 067 females, with a median age of 18 years. Totally 3 053 skin and soft tissue expansion procedures were performed with 4 266 expanders implanted. The following indexes were selected as predictor variables, including patients' age, gender, marital status, ethnicity, hospital admission, surgical indication, disease duration, with/without history of smoking, history of drinking, history of blood transfusion, history of underlying diseases, and inability to use cephalosporin antibiotics due to allergy, number of expander in a single placement, rated volume of expander, water injection rate of expander in the first time, placement site of expander, anesthesia method, duration of operation, and with/without postoperative hematoma evacuation, and infection at the placement site of expander as the outcome variable. Univariate analysis of the data was performed using least absolute shrinkage and selection operator (LASSO) regression to screen the potential risk factors affecting infection at the placement sites of expanders, the factors selected by the univariate analysis were subjected to binary multivariate logistic regression analysis to screen the independent risk factors affecting infection at the placement sites of expanders, and a nomogram prediction model for the occurrence of infection at the placement sites of expanders was established. The C index and Hosmer-Lemeshow goodness of fit test were used to evaluate the discrimination and accuracy of the model, respectively, and the bootstrap resampling was used for internal verification. Results The results of LASSO regression showed that age, gender, hospital admission, surgical indication, disease duration, history of drinking, history of heart disease, history of viral hepatitis, history of hypertension, inability to use cephalosporin antibiotics due to allergy, number of expander in a single placement, rated volume of expander, placement site of expander, postoperative hematoma evacuation were the potential risk factors for infection at the placement sites of expanders (regression coefficient= - 0.005, 0.170, 0.999, 0.054, 0.510, - 0.003, 0.395, - 0.218, 0.029, 0.848, - 0.116, 0.175, 0.085, 0.202). Binary multivariate logistic regression analysis showed that male, emergency admission, disease duration ≤1 year, inability to use cephalosporin antibiotics due to allergy, rated volumes of expanders ≥200 mL and <400 mL or ≥400 mL, and expanders placed in the trunk or the limbs were the independent risks factors for infection at the placement sites of expanders (odds ratio=1.37, 3.21, 2.00, 2.47, 1.70, 1.73, 1.67, 2.16, 95% confidence interval=1.04 - 1.82, 1.09 - 8.34, 1.38 - 2.86, 1.29 - 4.41, 1.07 - 2.73, 1.02 - 2.94, 1.09 - 2.58, 1.07 - 4.10, P<0.05 or P<0.01). The C index for evaluating the discriminative degree of the model was 0.63, the Hosmer-Lemeshow goodness of fit test for evaluating the accuracy of the model showed P=0.685, and the C index for internal validation by the bootstrap resampling was 0.60. Conclusions Male, emergency admission, disease duration ≤1 year, inability to use cephalosporin antibiotics due to allergy, rated volume of expander ≥200 mL, and expanders placed in the trunk or the limbs are the independent risk factors for infection at the placement sites of expanders. The clinical prediction model for infection risk at the placement sites of expanders was successfully established based on these factors and showed a certain predictive effect.

Effects and molecular mechanism of histone deacetylase 6 inhibitor Tubastatin A on the prolifera- tion and movement of human skin fibroblasts

Zhang Can, Zhang Qiong, Zhang Junhui, Wang Fan, Zhang Jiaping

2021, 37(9): 853-859. doi: 10.3760/cma.j.cn501120-20200519-00274

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Objective To explore the effects and possible molecular mechanism of histone deacetylase 6 (HDAC6) inhibitor Tubastatin A on the proliferation and movement of human skin fibroblasts (HSFs). Methods The experimental research method was used. HSFs in logarithmic growth phase were taken and divided into negative control group, 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group according to the random number table. The HSFs in negative control group were added with Dulbecco′s modified eagle medium with the final volume fraction of 0.1% dimethyl sulfoxide (hereinafter referred to as the complete medium), and the other three groups were added with the complete medium with the corresponding final molarity of Tubastatin A. After 24 h of conventional culture, the cell proliferation activity was detected using cell counting kit 8 (CCK-8) method and 5-ethynyl-2'-deoxyuridine (EdU) staining; the range of motion of cells within 3 h was observed under the living cell workstation, and the curve movement velocity of the cells was calculated. The protein expressions of extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2) were detected by Western blotting, and the ratio of p-ERK1/2 to ERK1/2 was calculated to represent the activity of ERK1/2. The sample number in cell proliferation activity detection with CCK-8 method was 6, while the sample numbers in other experiments were 3. Data were statistically analyzed with one-way analysis of variance and least significant difference test. Results After 24 h of culture, CCK-8 method and EdU staining showed that compared with negative control group, the cell proliferation activities in 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group were significantly decreased (P<0.01). After 24 h of culture, CCK-8 method showed that compared with 1 μmol/L Tubastatin A group, the cell proliferation activity in 10 μmol/L Tubastatin A group was significantly decreased (P<0.05); EdU staining showed that compared with 1 μmol/L Tubastatin A group, the cell proliferation activities in 5 μmol/L Tubastatin A group and 10 μmol/L Tubastatin A group were significantly decreased (P<0.05 or P<0.01). Within 3 h of observation, the ranges of cell motion in 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group were obviously reduced compared with that in negative control group. Within 3 h of observation, the curve movement velocity of cells in negative control group was (0.780±0.028) μm/min, which was obviously faster than (0.594±0.023), (0.469±0.028), and (0.391±0.021) μm/min of 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group (P<0.01); the curve movement velocity of cells in 1 μmol/L Tubastatin A group was obviously faster than those in 5 μmol/L Tubastatin A group and 10 μmol/L Tubastatin A group (P<0.01); the curve movement velocity of cells in 5 μmol/L Tubastatin A group was obviously faster than that in 10 μmol/L Tubastatin A group (P<0.05). After 24 h of culture, compared with negative control group, the activities of ERK1/2 of cells in 1 μmol/L Tubastatin A group, 5 μmol/L Tubastatin A group, and 10 μmol/L Tubastatin A group were decreased significantly (P<0.01); compared with 1 μmol/L Tubastatin A group, the activities of ERK1/2 of cells in 5 μmol/L Tubastatin A group and 10 μmol/L Tubastatin A group were decreased significantly (P<0.01); compared with 5 μmol/L Tubastatin A group, the activity of ERK1/2 of cells in 10 μmol/L Tubastatin A group was decreased significantly (P<0.05). Conclusions HDAC6 inhibitor Tubastatin A may mediate the inhibitory effect on proliferation and movement of HSFs by inhibiting the activity of ERK1/2.

Effects and mechanism of hepatocyte growth factor-modified human adipose mesenchymal stem cells on wound healing of full-thickness skin defects in diabetic rats

Zhang Yue, Han Fei, He Ting, Ji Peng, Zhang Zhi, Tao Ke

2021, 37(9): 860-868. doi: 10.3760/cma.j.cn501120-20200626-00329

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Objective To investigate the effects and mechanism of hepatocyte growth factor (HGF)-modified human adipose mesenchymal stem cells (ADSCs) on the wound healing of full-thickness skin defects in diabetic rats. Methods The experimental research method was adopted. The discarded abdominal adipose tissue was collected from a 35-year-old healthy female who underwent abdominal liposuction in the Department of Plastic Surgery of the First Affiliated Hospital of Air Force Medical University in December 2019. The long spindle-shaped primary ADSCs were obtained by collagenase digestion, and the third passage of cells were identified by flow cytometry to positively express ADSCs surface markers CD29 and CD90 and negatively express CD34 and CD45. The third passage of ADSCs were used for the subsequent experiments. ADSCs were transfected with lentivirus-mediated HGF for 4 h (obtaining HGF modified ADSCs) and then routinely cultured for 24 h. The cell morphology was observed under an inverted phase contrast microscope, and the transfection rate was calculated. Eighty-one male Sprague-Dawley rats aged 4 weeks were induced into diabetic rat model by high glucose and high fat diet combined with streptozotocin injection. A full-thickness skin defect wound of 1.5 cm×1.5 cm was made on the back of each rat. The injured rats were divided into phosphate buffer solution (PBS) group, ADSCs alone group, and HGF-modified ADSCs group according to the random number table, with 27 rats in each group. The rats were injected with the same volume of corresponding substances around the wound on post injury day (PID) 1, 3, and 7, respectively. Nine rats in each group were selected according to the random number table, the wound area of whom was measured on PID 0 (immediately), 3, 7, 10, and 14 (after injection on injection day), and the wound healing rates on PID 3, 7, 10, and 14 were calculated. Nine remaining rats in each group were sacrificed after injection on PID 3 and 7, respectively, and the skin tissue around the wound were collected. The mRNA expressions of inflammatory factors such as tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), and IL-10 on PID 3 and collagen type Ⅰ and Ⅲ on PID 7 were detected by real-time fluorescent quantitative reverse transcription polymerase chain reaction. The expression level of vascular endothelial growth factor (VEGF) was detected by enzyme-linked immunosorbent assay on PID 7. The protein expression of nuclear factor κb-p65 on PID 3 and phosphorylation level of protein kinase B (Akt) on PID 7 were detected by Western blotting. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, least significant difference t test, and Bonferroni correction. Results After 24 h of culture, the HGF-transfected human ADSCs showed good morphology, which was not different with the non-transfected ADSCs, and the transfection rate reached 90%. On PID 3, 7, 10, and 14, the wound healing rates of rats in HGF-modified ADSCs group were (31.5±1.0)%, (75.2±2.0)%, (92.2±1.3)%, and (99.1±1.8)%, respectively, being significantly higher than (21.4±1.3)%, (61.4±1.5)%, (80.1±2.1)%, and (92.4±1.8)% in PBS group and (25.1±2.1)%, (67.2±1.3)%, (89.3±1.4)%, and (95.1±2.1)% in ADSCs alone group (t=1.452, 0.393, 0.436, 0.211, 4.982, 3.011, 4.211, 7.503, P<0.05 or P<0.01). On PID 3, compared with those in PBS group and ADSCs alone group, the mRNA expressions of TNF-α and IL-1β and protein expression of nuclear factor κb-p65 in the skin tissue around the wound of rats in HGF-modified ADSCs group were significantly decreased (t=7.281, 17.700, 9.447, 6.231, 13.083, 7.783, P<0.01), and the mRNA expression of IL-10 in the skin tissue around the wound of rats in HGF-modified ADSCs group was significantly increased (t=-6.644, -6.381, P<0.01). On PID 7, compared with those in PBS group and ADSCs alone group, the mRNA expressions of collagen type Ⅰ and Ⅲ, the expression level of VEGF, and the phosphorylation level of Akt in the skin tissue around the wound of rats in HGF-modified ADSCs group were significantly increased (t=-5.126, -4.347, -5.058, -3.367, -10.694, -19.876, -4.890, -6.819, P<0.05 or P<0.01). Conclusions HGF-modified human ADSCs can significantly promote the wound healing of full-thickness skin defects in diabetic rats. The mechanism may be related to the inhibition of TNF-α and IL-1β expression, the promotion of IL-10, collagen type Ⅰ and Ⅲ, and VEGF expression, which could be related to the inhibition of nuclear factor κB signaling pathway, and the promotion of Akt signaling pathway.

Effects and mechanism of estrogen receptor β agonist on the migration and oxidative stress of human umbilical vein endothelial cell under high glucose condition

Guo Xiaoyu, Zhou Xueqing, Xie Weiguo

2021, 37(9): 869-874. doi: 10.3760/cma.j.cn501120-20200720-00352

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Objective To investigate the effects and related mechanism of estrogen receptor β (ERβ) agonist on the migration and oxidative stress of human umbilial vein endothelial cells (HUVECs) under high glucose condition. Methods The experimental research method was adopted. HUVECs were routinely cultured and passaged, and then cells of the logarithmic growth phase were collected for the subsequent experiments. The cells were divided into three groups according to the random number table, including normal control group (cultured with Roswell Park Memorial Institute 1640 cell culture medium (the same cell culture medium below) containing 5.5 mmol/L D-glucose), high glucose alone group (cultured with cell culture medium containing 25.0 mmol/L D-glucose alone), and high glucose+ERβ agonist diarylpropionitrile (DPN) group (cultured with cell culture medium containing 25.0 mmol/L D-glucose and 10 μmol/L DPN). Scratch test was conducted to detect the cell migration rate in the 3 groups at 24 h post scratching. Fluorescent probe method was used to detect the reactive oxygen species (ROS, denoted by red fluorescence intensity) of cells in the 3 groups on 5 d post culture. Western blotting was used to detect the protein expression levels of vascular endothelial growth factor (VEGF) and superoxide dismutase 2 (SOD2) of cells in the 3 groups on 5 d post culture. In the above-mentioned experiments, cells were grouped and cultured correspondingly as before, the number of samples in each group was 5. Data were statistically analyzed with one-way analysis of variance and least significant difference t test. Results At 24 h post scratching, the cell migration rate in high glucose alone group was (36±5)%, which was significantly lower than (76±4)% of normal control group and (65±5)% of high glucose+DPN group (t=14.511, 9.603, P<0.01), and the cell migration rate in high glucose+DPN group was significantly lower than that in normal control group (t=3.943, P<0.01). On 5 d post culture, the level of ROS of cells in high glucose alone group (1.81±0.12) was significantly increased compared with normal control group and high glucose+DPN group (1.00±0.14, 0.91±0.15, t=9.679, 10.549, P<0.01), while the level of ROS of cells in normal control group and high glucose+DPN group were close (t=1.031, P>0.05). On 5 d post culture, the protein expression levels of VEGF and SOD2 of cells in high glucose alone group were significantly lower than the levels of normal control group (t=14.175, 13.787, P<0.01) and high glucose+DPN group (t=6.321, 17.750, P<0.01). The protein expression level of VEGF of cells in high glucose+DPN group was significantly lower than the level of normal control group (t=7.206, P<0.05), while the protein expression level of SOD2 of cells in high glucose+DPN group was significantly higher than the level of normal control group (t=2.890, P<0.05). Conclusions The activation of ERβ can improve the inhibition of HUVECs migration induced by high glucose and alleviate oxidative stress injury induced by high glucose, which may be achieved by promoting the expression of VEGF and SOD2.

Clinical effect of perforator flap combined with toe transplantation for repairing thumb damage with soft tissue defect of hand

Yang Tao, Chen Jia, Cheng Beibei, Wang Shuai, Jiang Xiaomeng, Song Li, Zhou Mingwu, Li Shimin

2021, 37(9): 875-879. doi: 10.3760/cma.j.cn501120-20200727-00360

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Objective To investigate the clinical effect of perforator flap combined with toe transplantation for repairing thumb damage with soft tissue defect of hand. Methods The retrospective observational study method was used. From May 2014 to June 2019, 8 patients with thumb damage and soft tissue defect of hand were admitted to the 988th Hospital of Joint Logistic Support Force of PLA, including 6 males and 2 females, aged from 25 to 46 years. Among them, thumb damage in 3 cases were degree Ⅱ, 1 case was degree Ⅲ, and 4 cases were degree Ⅳ. All thumb damage were repaired with perforator flap combined with toe transplantation. The skin and soft tissue defects of hand were repaired by free anterolateral thigh perforator flap in 6 cases and free deep inferior epigastric perforator flap in 2 cases. The thumb damage of degree Ⅱ was repaired by the first toe transplantation combined with perforator flap, and thumb damage of degree Ⅲ or Ⅳ was repaired by the second toe transplantation combined with perforator flap. The survival and blood supply of reconstructed thumbs and flaps, and wound healing of donor sites were observed after surgery. All the patients were followed up for 10 to 18 months, the appearance of the reconstructed thumbs, sensory recovery, and foot walking function were observed. At the final follow-up, the functional reconstruction of the thumb was evaluated. Results All the blood supply and survival of the reconstructed thumbs and flaps were good, and all the wounds of donor sites healed well. During the follow-up, the appearances of the reconstructed thumb and flap were good, the sensation of pain and touch of the finger pulp recovered well, and no significant impact on foot walking function was observed. At the final follow-up, the function of reconstructed thumb was evaluated as excellent in 4 cases, good in 3 cases, and fair in 1 case. Conclusions The repair method of perforator flap combined with toe transplantation technique can complete the targeted repair of thumb damage with skin and soft tissue defect of hand in one stage, minimizing the foot donor site injury and shortening the course of disease and early rehabilitation, which is one of the ideal methods for the treatment of complex thumb damage.

Research advances on early enteral nutritional therapy in severe burn patients

Luo Yue, Li Ning

2021, 37(9): 880-884. doi: 10.3760/cma.j.cn501120-20210621-00223

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Severe burns can lead to persistent hypermetabolism. Studies have shown that early enteral nutrition therapy is related to improving the prognosis of severe burn patients, reducing the mortality rate, and reducing gastrointestinal complications and infectious complications. This article introduces the nutritional benefits and non-nutritive benefits of early enteral nutrition therapy for severe burn patients (maintaining the integrity of the intestinal mucosa function and structure and promoting wound healing), the timing and pathways of nutritional intervention, ways of energy evaluation, and nutritional monitoring, etc., aiming to provide a reference for the clinical decision-making of early enteral nutrition therapy.

Research advances on the application of biocompatible materials in treating diabetic wounds

Liu Kaituo, Hu Dahai

2021, 37(9): 885-886. doi: 10.3760/cma.j.cn501120-20200619-00316

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At present, a variety of biocompatible materials have been applied in treating diabetic wounds, and some biocompatible materials have played an important role. Although biocompatible materials have advantages such as decreasing exposure and tension of wounds, and providing microenvironment to stimulate cell proliferation and migration, they also have disadvantages such as unstable effects and unclear mechanism. Therefore, this article reviews the recent advances on the biocompatible materials for the treatment of diabetic wounds, and discusses the possible development directions in the field of biocompatible materials.

Research advances on the cholinergic inflammatory reflex and inflammation resolution

Chen Zhuo, He Xiao, Yao Mengwei, Li Zhan, Xu Xiang

2021, 37(9): 885-894. doi: 10.3760/cma.j.cn501120-20200609-00299

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The vagus nerve plays an important role in regulating the homeostasis of inflammation. Inflammation signals in the body are passed to the vagus nerve efferent fibers via nerve reflexes, and the signals generated by efferent fibers will play an anti-inflammatory role in various inflammatory diseases through immune cells such as T cells that express choline acetyltransferase and macrophages. However, the resolution of inflammation is not only the interaction between pro-inflammatory and anti-inflammatory cytokines, but also an active process of biosynthesis, including the synthesis of various pro-resolving mediators and their physiological utility process. Moreover, the cholinergic inflammation reflex also plays a crucial role in inflammation resolution. This review reviews and summarizes the cholinergic inflammatory reflex and its key role in the process of inflammation resolution.

Research advances on the effect of resistance training on the rehabilitation of burned children

Yang Sha, Qiu Lin

2021, 37(9): 895-899. doi: 10.3760/cma.j.cn501120-20200717-00349

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Children are at a critical period of growth and development, their body function and quality of life will be greatly reduced by the direct or indirect impact of burns. Therefore, rehabilitation therapy after burns is very important for the treatment of burned children. Resistance training is one of the rehabilitation exercises for patients. This article reviews the effects of resistance training on the rehabilitation of burned children, summarizes the advantages and disadvantages of the current related studies, and provides references and research approach for future studies, so as to improve the overall prognosis of burned children.

Research advances on skin sweat gland regeneration induced by stem cells and tissue engineering

Zeng Yingnan, Kang Yangbo, Xu Yong'an

2021, 37(9): 900-904. doi: 10.3760/cma.j.cn501120-20200624-00328

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As the largest organ in mammals, skin is the first protective barrier against external stimuli. Sweat glands are one of the important cutaneous appendages and play an important role in maintaining electrolyte balance and regulating body temperature. Patients with extensive deep burns usually suffer from damage to deep dermis or the entire skin layer. The damaged sweat glands are difficult to repair and even unable to regenerate, which seriously affects patients' sweating and thermo-regulation function, reduces patients' quality of lives. How to achieve the functionalization of sweat glands has become one of the important researches in regenerative medicine of damaged skin. This review summarizes the translational and application technology of sweat gland regeneration and repair using stem cells and tissue engineering, in order to provide a theoretical basis for the research of sweat gland regeneration.

2021 Vol. 37, No. 9

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