OBJECTIVE To establish the ultra-high performance liquid chromatography （UPLC） fingerprint of Jingtian granule， and to evaluate its quality by chemical pattern recognition. METHODS Luna® Omega Polar C18 column （150 mm×2.1 mm， 1.6 μm） was used as the chromatographic column， and acetonitrile-0.2% phosphoric acid solution was used as the mobile phase for gradient elution. The flow rate was 0.2 mL/min， the column temperature was 30 ℃， and the detection wavelength was 265 nm. With peak 16 as the reference peak， the UPLC fingerprint of Jingtian granule was established by the Similarity Evaluation System of Chromatographic Fingerprint of Traditional Chinese Medicine （2012 edition）. The common peaks were identified， the similarity evaluation was carried out， and the ownership of each common peak was confirmed. Hierarchical cluster analysis （HCA） and principal component analysis （PCA） in chemical pattern recognition methods were used to classify 13 batches of samples （S1- S13）， and orthogonal partial least squares-discriminant analysis （OPLS-DA） was used to identify the key components of the differences between different batches of samples. RESULTS RSDs of precision， repeatability and stability of the UPLC method were not more than 4.4%. A total of 25 common peaks were identified in the fingerprints of 13 batches of Jingtian granules. By comparing with the reference substance fingerprint， 10 common peaks were identified， namely peak 3 （hydroxymethyl-2-furaldehyde）， peak 5 （salidroside）， peak 8（chlorogenic acid）， peak 15 （cinnamic acid）， peak 19 （aloe-emodin）， peak 20 （ammonium glycyrrhizinate）， peak 21 （rhein）， peak 23 （emodin）， peak 24 （glycyrrhetinic acid）， peak 25 （chrysophanol）. The similarities of fingerprints of 13 batches of samples were 0.955-0.996. The results of HCA showed that 13 batches of samples could be divided into three categories， among which samples S1， S5， S7， S11-S13 were clustered in one category， S4 and S6 were clustered in one category， S2， S3 and S8-S10 were clustered in one category. PCA results showed that the cumulative variance contribution rate of principal components 1-7 was 92.666%. OPLS-DA further identified 13 differential components， which were mainly derived from Polygonati Rhizoma with wine steaming， Rhodiolae Crenulatae Radix Et Rhizoma， prepared Rhei Radix Et Rhizoma and Glycyrrhizae Radix Et Rhizome Praeparata Cum Melle. CONCLUSIONS The established UPLC fingerprint of Jingtian granule is simple， stable and reproducible. Combined with the chemical pattern recognition method， it can effectively reveal the overall quality difference between different batches of Jingtian granule. The quality of Polygonati Rhizoma with wine steaming， Rhodiolae Crenulatae Radix Et Rhizoma， prepared Rhei Radix Et Rhizoma， Dioscoreae Nipponicae Rhizoma， Polyporus， Cinnamomi Ramulus， Glycyrrhizae Radix Et Rhizome Praeparata Cum Melle is the key to the overall quality of Jingtian granule.

OBJECTIVE: To assess the value of single sperm sequencing in preimplantation genetic testing for monogenic disease (PGT-M). METHODS: A Chinese couple with two children whom had died of Spinal muscular atrophy (SMA) and attended the Jiangxi Provincial Maternal and Child Health Care Hospital in June 2020 was selected as the subject. Eleven single sperm samples were isolated by mechanical immobilization and subjected to whole genome amplification. Real-time PCR and Sanger sequencing were used to detect the SMN1 variants in the single sperm samples. Genomic DNA of the wife, her parents and the husband, as well as one single sperm sample harboring the SMN1 variant and two single sperm samples without the variant were used for the linkage analysis. Targeted capture and high-throughput sequencing were carried out to test 100 single nucleotide polymorphisms distributed within 2 Mb up- and downstream the variant site. The haplotypes linked with the SMN1 variants were determined by linkage analysis. Blastocyst embryos were harvested after fertilizing by intracytoplasmic sperm injection. Cells from the trophoblasts of each embryo were biopsied and subjected to whole genome amplification and targeted capture and high-throughput sequencing to determine their carrier status. Chromosomal aneuploidy of wild-type embryos was excluded. An euploid embryo of high quality was transferred. Amniotic fluid sample was taken at 18 weeks of gestation to confirm the status of the fetus. RESULTS: Genetic testing showed that the couple both had deletion of exons 7 ~ 8 of the SMN1 gene. The wife has inherited the deletion from her father, while the husband was de novo. The haplotypes of the husband were successfully constructed by single sperm sequencing. Preimplantation genetic testing has indicated that 5 embryos had harbored the heterozygous variant, 4 embryos were of the wild type, among which 3 were euploid. Prenatal diagnosis during the second trimester of pregnancy has confirmed that the fetus did not carry the deletion. CONCLUSION By single sperm sequencing and PGT-M, the birth of further affected child has been successfully avoided.
Humans ; Pregnancy ; Female ; Child ; Male ; Preimplantation Diagnosis ; East Asian People ; Semen ; Genetic Testing ; Muscular Atrophy, Spinal/genetics* ; Aneuploidy ; Blastocyst/pathology* ; High-Throughput Nucleotide Sequencing ; Spermatozoa

Objective:To analyze the risk factors of early myocardial injury and the impact of early myocardial injury on prognosis of patients with extensive burns.Methods:A retrospective case series study was conducted. From January 2018 to August 2022, 361 patients with extensive burns who met the inclusion criteria were admitted to Tongren Hospital of Wuhan University & Wuhan Third Hospital, including 231 males and 130 females, aged 50 (36, 58) years, with total burn area of 45% (35%, 60%) total body surface area. According to the highest level of creatine kinase isoenzyme-MB (CK-MB) within 72 h post injury, the patients were divided into early myocardial injury group (CK-MB≥75 U/L, 182 patients) and non-early myocardial injury group (CK-MB<75 U/L, 179 patients). The following data of patients in the 2 groups were collected and analyzed, including gender, age, total burn area, admission time post injury, combination with shock on admission, combination with inhalation injury on admission; the main blood test indexes such as myocardial enzyme spectrum, blood routine, liver and kidney function, and electrolytes within 72 h post injury; and treatment outcomes and fatality rate. Data were statistically analyzed with chi-square test, independent sample t test, or Mann-Whitney U test. The multivariate logistic regression analysis was conducted to screen the independent risk factors for early myocardial injury and for death in patients with extensive burns. Results:There were statistically significant differences in gender, combination with shock on admission, total burn area, and admission time post injury of patients between the two groups (with χ2 values of 6.40 and 6.10, Z values of 5.41 and 3.03, respectively, P<0.05). There were no statistically significant differences in age, combination with inhalation injury on admission of patients between the two groups ( P>0.05). The CK-MB, creatine kinase, lactate dehydrogenase, α-hydroxybutyrate dehydrogenase, white blood cell count, neutrophil-to-lymphocyte ratio (NLR), alanine aminotransferase (ALT), aspartate aminotransferase, potassium, and hemoglobin within 72 h post injury were significantly higher than those in non-early myocardial injury group (with Z values of 15.40, 6.26, 7.59, 7.02, 2.64, 4.53, 4.07, 6.32, and 4.12, t=2.34, respectively, P<0.05), while the level of calcium was significantly lower than that in non-early myocardial injury group ( Z=2.72, P<0.05). There were no statistically significant differences in other blood test indexes of patients between the two groups ( P>0.05). The total burn area, admission time post injury, NLR and ALT within 72 h post injury were the independent risk factors for early myocardial injury in patients with extensive burns (with odds ratios of 1.03, 1.07, 1.04, and 1.02, 95% confidence intervals of 1.02-1.05, 1.00-1.11, 1.02-1.07, and 1.00-1.03, respectively, P<0.05). The fatality rate of patients in early myocardial injury group was 8.8% (16/182), which was significantly higher than 2.8% (5/179) in non-early myocardial injury group ( χ2 =5.93, P<0.05). Early myocardial injury, age, combination with shock on admission, and combination with inhalation injury on admission were the independent risk factors for death in patients with extensive burns (with odds ratios of 3.60, 1.04, 6.53, and 3.14, 95% confidence intervals of 1.17-11.05, 1.01-1.07, 1.39-30.68, and 1.15-8.56, respectively, P<0.05). Conclusions:The total burn area, admission time post injury, NLR and ALT within 72 h post injury were the independent risk factors for early myocardial injury in patients with extensive burns. Patients with extensive burns with early myocardial injury have a higher fatality rate, and early myocardial injury is an independent risk factor for the patients' death.

Objective:To explore the biological role and clinical significance of ubiquitin-specific protease 7 (USP7) in the carcinogenesis of scar ulcer.Methods:A retrospective observational study combined with bioinformatics analysis was used. The RNA expression profile data of USP7 in tumor and/or its corresponding paracancular normal tissue were obtained from The Cancer Genome Atlas (TCGA) database and the Gene Expression Omnibus database, and the RNA sequencing data were transformed by log 2. The variations of USP7 gene were analyzed by cBioPortal database. The USP7 mRNA expression in tumor and adjacent normal tissue in TCGA database were obtained by using the "Gene_DE" module in TIMER 2.0 database. The survival rates of patients with high and low USP7 expression in cutaneous melanoma (SKCM), cervical squamous cell carcinoma (CESC), lung squamous cell carcinoma (LUSC), and head and neck squamous cell carcinoma (HNSC) were analyzed using the Gene Expression Profile Interactive Analysis 2 (GEPIA2) database, and the Kaplan-Meier survival curves were drawn. Sangerbox database was used to analyze the correlation of USP7 expression in pan-cancer with microsatellite instability (MSI) or tumor mutation burden (TMB) pan-cancer. Through the "correlation analysis" module in the GEPIA2 database, the correlation of USP7 expression in pan-cancer with the expression levels of five DNA mismatch repair genes ( MLH1, MSH2, MSH6, PMS2, and EPCAM) and three essential DNA methyltransferases (DNMT)--DNMT1, DNMT3A, and DNMT3B were evaluated. The USP7 expression in CESC, HNSC, LUSC, and SKCM and its correlation with infiltration of immune cells (B cells, CD4 + T cells, CD8 + T cells, neutrophils, macrophages, and dendritic cells) were analyzed by the "Immune-Gene" module in TIMER 2.0 database. The "Similar Genes Detection" module of GEPIA2 database was used to obtain the top 100 protein sets with similar expression patterns to USP7. Intersection analysis was performed between the aforementioned protein sets and the top 50 protein sets that were directly physically bound to USP7 obtained by using the STRING database. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment analysis were performed for the two protein sets mentioned above using the DAVID database. The samples of normal skin, hypertrophic scar, scar ulcer, and scar carcinoma with corresponding clinicopathologic features were collected from the Department of Pathology of Tongren Hospital of Wuhan University & Wuhan Third Hospital from October 2018 to October 2022, and the USP7 expression in tissue was detected by immunohistochemical method, with the number of samples of 6. Data were statistically analyzed with Log-rank test, one-way analysis of variance, and Bonferroni test. Results:In pan-cancer, the main gene variations of USP7 were mutation and amplification, and the top 3 tumors with the highest variation frequency (>6%) were bladder urothelial carcinoma, SKCM, and endometrial carcinoma. The main mutation of USP7 gene in pan-cancer was missense mutation. In SKCM with the highest mutation frequency, the main type of mutation was missense mutation in USP7_ICP0_bdg domain. USP7 mRNA expression in breast invasive carcinoma, bile duct carcinoma, colon carcinoma, esophageal carcinoma, HNSC, renal chromophobe cell carcinoma, hepatocellular carcinoma, lung adenocarcinoma, LUSC, prostate carcinoma, and gastric carcinoma was significantly higher than that in corresponding paracancer normal tissue ( P<0.05). USP7 mRNA expression in glioblastoma multiforme, renal clear cell carcinoma, renal papillary cell carcinoma, and thyroid carcinoma was significantly lower than that in corresponding paracancular normal tissue ( P<0.05). In addition, USP7 mRNA expression in SKCM metastases was much higher than that in primary tumor tissue ( P<0.05). Survival curves showed no significant difference in survival rate between patients with high USP7 expression and patients with low USP7 expression in CESC, HNSC, LUSC, and SKCM (Log-rank P>0.05, with hazard ratios of 1.00, 0.99, 1.00, and 1.30, respectively). USP7 expression in colon cancer, colorectal cancer, thymic cancer, and thyroid cancer was negatively correlated with TMB (with Pearson correlation coefficients of -0.26, -0.19, -0.19, and 0.11, respectively, P<0.05). USP7 expression in glioma, CESC, lung adenocarcinoma, mixed renal carcinoma, and LUSC was positively correlated with MSI expression (with Pearson correlation coefficients of 0.22, 0.14, 0.15, 0.08, and 0.14, respectively, P<0.05), and USP7 expression in colon cancer, colorectal cancer, invasive breast cancer, prostate cancer, HNSC, thyroid cancer, and diffuse large B-cell lymphoma were significantly negatively correlated with MSI expression (with Pearson correlation coefficients of -0.31, -0.27, -0.13, -0.19, -0.16, -0.18, and -0.53, respectively, P<0.05). The expression of USP7 in CESC was positively correlated with that of both MSH2 and MSH6 (with Spearman correlation coefficients of 0.51 and 0.44, respectively, P<0.05), and the expression of USP7 in HNSC was positively correlated with the expression of EPCAM, MLH1, MSH2, MSH6, and PMS2 (with Spearman correlation coefficients of 0.39, 0.14, 0.49, 0.54, and 0.41, respectively, P<0.05), and the expression of USP7 in LUSC was positively correlated with the expression of EPCAM, MSH2, MSH6, and PMS2 (with Spearman correlation coefficients of 0.20, 0.36, 0.40, and 0.34, respectively, P<0.05), and the expression of USP7 in SKCM was positively correlated with the expression of EPCAM, MLH1, MSH2, MSH6, and PMS2 (with Spearman correlation coefficients of 0.11, 0.33, 0.42, 0.55, and 0.34, respectively, P<0.05). The expression of USP7 in CESC, HNSC, LUSC, and SKCM was significantly positively correlated with the expression of DNMT1, DNMT3A, and DNMT3B (with Spearman correlation coefficients of 0.42, 0.34, 0.22, 0.45, 0.52, 0.22, 0.36, 0.36, 0.22, 0.38, 0.46, and 0.21, respectively, P<0.05). The expression of USP7 in CESC, HNSC, LUSC, and SKCM was positively correlated with CD4 + T cell infiltration (with Partial correlation coefficients of 0.14, 0.22, 0.13, and 0.16, respectively, P<0.05). Being similar to the pattern of USP7 expression and ranked among top 100 protein sets, the top 5 proteins were C16orf72, BCLAF1, UBN, GSPT1, ERI2 (with Spearman correlation coefficients of 0.83, 0.74, 0.73, and 0.72, respectively, all P values<0.05). The top 50 protein sets that directly physically bind to USP7 overlapped with the aforementioned protein set by only one protein, thyroid hormone receptor interaction factor 12. KEGG enrichment analysis showed that USP7 related genes were involved in cell cycle, spliceosome, cell senescence, and p53 signal pathway. GO enrichment analysis showed that USP7 related genes were involved in transcriptional regulation, protein ubiquitination, DNA repair, and cytoplasmic pattern recognition receptor signal pathways. Analysis of clinical samples showed that USP7 expression was significantly higher in hypertrophic scars (0.35±0.05), scar ulcers (0.43±0.04), and scar cancers (0.61±0.03) than in normal skin (0.18±0.04), P<0.05. Conclusions:USP7 may be a clinical biomarker for the progression of cicatricial ulcer cancer.

On August 6, 2015, a male infant with ectodermal dysplasia/skin fragility syndrome at 6 hours of birth was admitted to the Burn Department of Tongren Hospital of Wuhan University & Wuhan Third Hospital. The ulcerous skin tissue in thoracic area was harvested. The histopathological change of wound tissue was observed with hematoxylin-eosin staining. The result showed that the epidermal muscle cell layer was slightly released, there were bullae under the epidermis, the dermal papilla layer disappeared, and a small amount of inflammatory cells infiltrated in the dermis. The expression of plakophilin 1 (PKP1) in wound tissue was observed with immunohistochemical staining. The result showed that the PKP1 expression was completely absent. The PKP1 gene mutation site was identified by target sequencing. The result showed that the PKP1 gene had a homozygous mutation at intron ( PKP1: c.203-1G>A). Most of the wounds of the pediatric patient healed after 35 days of treatment, with many scattered residual wounds visible, and new blisters and skin lesions continue to appear.

Objective:To investigate the causes of death and etiological characteristics of skin tissue donors, and to provide reference for allogeneic skin transplantation.Methods:From October 2008 to October 2018, 49 skin tissue donors accepted by the Burn Department of Wuhan Third Hospital met the inclusion criteria of this study, and a cross-sectional study was conducted. According to the cause of death, the donors were divided into accidental death group (19 cases) and non-accidental death group (30 cases). The sex and death age of 49 donors were recorded, and the death age between different sex donors and that of donors between accidental death group and non-accidental death group were compared. Diseases or circumstances that caused the death of donors, hepatitis B, hepatitis C, acquired immunodeficiency syndrome, syphilis virus carrying status, and peripheral blood microbial culture results of 49 donors were recorded, and the detection of blood-borne infectious risk factors of donors between accidental death group and non-accidental death group was compared. Abnormal skin tissue was also selected during allogenic skin graft preparing for pathological examination. Data were statistically analyzed with Mann-Whitney U test and continuity correction chi-square test. Results:(1) Out of the 49 donors in this group, 38 were male (77.55%) and 11 were female (22.45%). The death age was 42.00 (24.00, 55.00) years, and the death age of male donors was similar to that of female donors ( Z=0.120, P>0.05). The death age of donors in accidental death group was lower than that in non-accidental death group, but the difference was not statistically significant ( Z=-1.581, P>0.05). (2) Among the causes and circumstances of the 49 donors in this group, there were 19 cases (38.78%) of injury, poisoning, and some other consequences of external causes, 11 cases (22.45%) of circulatory system diseases, 9 cases (18.37%) of tumors, 3 cases (6.12%) of nervous system diseases, 2 cases (4.08%) of respiratory system diseases, and 2 cases (4.08%) of congenital malformation, deformation, and chromosome abnormality, 1 case (2.04%) of blood and hematopoietic organ diseases and some diseases related to immune mechanism, 1 case (2.04%) of digestive system disease, and 1 case (2.04%) of genitourinary system disease. (3) There were 9 donors (18.37%) with blood-borne infectious risk factors among the 49 donors in this group, including 8 cases (16.33%) of blood-borne infectious diseases, which were 5 cases (10.20%) of hepatitis B, 2 cases (4.08%) of syphilis, and 1 case (2.04%) of hepatitis C, respectively. Blood microorganism culture was positive in 1 case (2.04%), in which multi-drug resistant Pseudomonas aeruginosa was detected. Risk factors of blood-borne infection were detected in 2 donors in accidental death group, with detection ratio lower than that in non-accidental death group (7 cases), but the difference was not statistically significant ( χ2=0.562, P>0.05). (4) A total of 8 donors′ abnormal skin tissue were selected, including 4 cases of intradermal pigmented nevus, 1 case of scar, 1 case of pseudoepithelioma hyperplasia, 1 case of epidermal verrucous hyperplasia, and 1 case of large amount of pigment granules in dermis. Conclusions:Non-accidental death caused by diseases is the main cause of death of skin tissue donors, and the risk of donor-derived infection of non-accidentally dead donors is slightly higher than that of accidentally dead donors. Before the allogeneic skin is obtained and transplanted, the cause of death of the donor should be carefully investigated, and the health status should be evaluated, so as to avoid the occurrence of donor-derived infection.

Objective To understand the correlation between health beliefs and family environment of stroke patients. Methods A questionnaire survey was carried out on 115 stroke patients with the first onset of stroke by using the special health belief simple table (SF-HBMS) and the family environment scale (Chinese version FES-CV), and the correlation was analyzed. The scores of each subscale of the family environment were compared with the domestic norm. Results The total score of health belief (75.15 ± 10.20) was at the middle level. There were significant differences in age (F=8.41), education level (F=4.44), complications (F=4.05), family history (t=2.68) and first visit time (F=3.76) among different characteristics of health belief scores (P < 0.01 or 0.05). The score of intimacy (6.23 ± 1.27) in family environment, emotional expression score (5.30 ± 1.97), success score (5.88 ±1.62), cultural score (4.54 ± 2.20) and organizational score (5.60 ±1.67) were all lower than the domestic norm and spear. The score of shield score (3.16 ± 2.00) was higher than that of domestic norm (P<0.01 or 0.05), and the total score of health belief was positively correlated with family intimacy (r=0.190), emotional expression (r=0.204), culture (r=0.206) and tissue (r=0.227) (P<0.05), and was negatively correlated with the contradiction (r=-0.186, P<0.05); regression analysis, whether there were family history (β=0.338, P<0.01), first onset time (β=0.242, P<0.01), family intimacy (β=1.614, P<0.05), emotional expression (β=1.114, P<0.05) were the factors affecting the health belief level of first stroke patients. Conclusions The level of health belief is closely related to family environment. It is suggested that the clinical medical staff should pay attention to the negative emotion and family psychological intervention, provide psychological support for the patients and their families, promote the promotion of their health beliefs, and reduce the rate of recurrence and disability.

Objective To observe the curative effect of clinically equivalent doses of Xuesaitong and ginaton injections on cerebral ischemia reperfusion (I/R) injury of rats.Methods Male rats were randomly divided into five groups:normal control group,sham-operation group,model control group,Xuesaitong group and ginaton group.The cerebral ischemia rat model was established by middle cerebral artery occlusion (MCAO).Rats in the Xuesaitong group were given 20 mg·kg-1 of Xuesaitong injection,and rats in the ginaton group were intravenously injected with 7.5 mg· kg-1of ginaton immediately after I/R injury and once daily for 7 days.Rats in the sham-operation group and model control group were given the same volume of 0.9％ sodium chloride solution.The score of ethology,volume of cerebral infarction,mortality,superoxide dismutase (SOD),malondialdehyde (MDA),xanthine oxidase (XOD),nitrogen oxide (NO) and NO synthase (NOS) in seruu were examined.Results Compared with model control group,Xuesaitong and ginaton effectively reduced behavioral score 96 h (P ＜ 0.05),120 h (P＜0.01),144 h (P＜0.01) and 168 h (P＜0.01) after I/R injury,the volume of cerebral infarction 168 h after I/R injury and NO content (P ＜ 0.05).But they had no effects on NOS,SOD,MDA,and XOD contents.Conclusion Curatively injecting Xuesaitong and ginaton can effectively reduce cerebral I/R injury,but no significant difference in curative efficacy is observed between Xuesaitong and ginaton at clinically equivalent doses.

Objective: To explore differential expression of microRNAs in serum of patients with severe burn and analysis of the signaling pathway at early stage. Methods: In this study, we included three healthy adult volunteers and three patients with severe burn, conforming to the inclusion criteria and hospitalized in Tongren Hospital of Wuhan University & Wuhan Third Hospital in July 2015. Venous whole blood of 6 mL of each burn patient and healthy volunteer was collected at 24 to 48 h post injury of burn patients. The whole blood was divided into burn group and healthy control group. Whole blood of 2 mL of each one was used to determine white blood cell count and neutrophile granulocyte content. Serum was separated from the other whole blood of 4 mL of each one. Half of serum was used to determine content of blood glucose, total protein, and albumin; another half of serum was used to extract total RNA with Trizol method. The differentially expressed microRNA, with differential expression ratio larger than or equal to 1.500 between 2 groups, were screened by microRNA chip technique. Then cluster analysis and functional enrichment analysis of Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway were performed on the differentially expressed microRNAs. Data were processed with t test. Results: (1) Content of white blood cell count, neutrophile granulocyte of whole blood, and blood glucose of serum of patients in burn group was obviously higher than that in healthy control group (with t values from 4.27 to 7.83, P<0.05 or P<0.01). Content of total protein and albumin of serum of patients in burn group was significantly lower than that in healthy control group (with t values respectively -12.80 and -12.36, P values below 0.01). (2)Compared with those in serum of healthy control group, differential expression ratios of 48 microRNAs in serum of burn group were larger than 1.500, with 22 up-regulated microRNAs and 26 down-regulated microRNAs. MicroRNA expression profile in serum of burn group was different from that of healthy control group. (3)Functional enrichment analysis of KEGG signaling pathway showed that compared with those in serum of healthy control group, microRNAs of differential expression in serum of burn group took part in tumor transcription misregulation signaling pathway, tumor proteoglycan signaling pathway, long-term potentiation signaling pathway, tumor associated microRNAs signaling pathway, citrate cycle signaling pathway, tumor necrosis factor signaling pathway, focal adhesion signaling pathway, endocytosis signaling pathway, insulin secretion signaling pathway, and estrogen signaling pathway. Conclusions MicroRNA expression profile in serum of patients with severe burn is different from that in serum of healthy adults. MicroRNAs of differential expression may take part in important pathophysiological process of energy metabolism, inflammatory response, and regulation of blood glucose at early stage of severe burn.

OBJECTIVETo observe the changes in the expressions of microRNA-126 in myocardial tissue and cardiac troponin I (cTnI) in serum of rats in the early stage of severe burn injury with analysis of their relationship, and to validate the relationship between microRNA-126 and myocardial damage in cellular level.

METHODS(1) Forty-eight SD rats were divided into sham injury group (n=8, without fluid therapy after sham injury) and burn injury group (n=40, inflicted with 30% TBSA full-thickness scald on the back, hereinafter referred to as burn, and received intraperitoneally injection of lactic acid Ringer's solution) according to the random number table. Blood was collected from abdominal aorta of rats in sham injury group at post injury hour (PIH) 1, and then these 8 rats were sacrificed for obtaining left ventricular tissue. Blood was respectively collected from abdominal aorta of 8 rats in burn injury group at PIH 3, 6, 12, 24, and 48, and then they were sacrificed and the left ventricular tissue was obtained at each time point. The expression of microRNA-126 in myocardial tissue was assessed by real-time fluorescent quantitative RT-PCR. Serum level of cTnI was assessed by ELISA. (2) Rat myocardial cell line H9C2 was divided into normal control group (NC, routinely cultured), stimulation group (S), negative transfection+stimulation group (NT+S), and transfection+stimulation group (T+S) according to the random number table. Cells in S group were treated with hypoxia for 24 h after being cultured with DMEM containing 10% burn serum obtained from rats in burn injury group at PIH 6 in experiment (1). Cells in NT+S group and T+S group were respectively transfected with the negative control of microRNA mimics and microRNA-126 mimics for 24 h, and then were given the same treatment as that of S group. The expression of microRNA-126 in myocardial cells was determined by real-time fluorescent quantitative RT-PCR (with the sample number of 3). Cell counting kit 8 was used to examine the vitality of myocardial cell (with the sample number of 4, denoted as absorbance value). Apoptotic rate of myocardial cells was determined by flow cytometer (with the sample number of 3). Data were processed with one-way analysis of variance and LSD-t test. The relationship between microRNA-126 expression in myocardial tissue and serum level of cTnI of rats was assessed by linear correlation analysis.

RESULTS(1) Compared with that of sham injury group at PIH 1, the expression levels of microRNA-126 in myocardial tissue of rats in burn injury group at PIH 3, 6, 12, 24, and 48 were significantly decreased (with t values from 5.68 to 9.79, P values below 0.01), reaching its nadir at PIH 24 (0.40 ± 0.08). Compared with that of sham injury group at PIH 1, the serum levels of cTnI of rats in burn injury group at PIH 3, 6, 12, 24, and 48 were significantly increased (with t values from 6.68 to 12.79, P values below 0.01), peaking at PIH 12 [(1 035 ± 177) pg/mL]. A significant negative correlation between the expression level of microRNA-126 in myocardial tissue and serum level of cTnI was observed in rats of burn injury group at each time point (r=-0.797, P<0.001). (2) Compared with those of NC group, the microRNA-126 expression levels in myocardial cells of S group and T+S group were respectively decreased and increased (with t values respectively 4.57 and 5.73, P<0.05 or P<0.01), the cell vitality levels were obviously decreased (with t values respectively 14.88 and 6.48, P values below 0.01), and the apoptotic rates were significantly increased (with t values respectively 13.82 and 6.96, P values below 0.01). Compared with that in NT+S group, the microRNA-126 expression level in myocardial cells of T+S group was significantly increased (t=6.77, P<0.01), the cell vitality level was obviously increased (t=8.23, P<0.001), and the apoptotic rate was significantly decreased (t=6.14, P<0.001).

CONCLUSIONSExpression level of microRNA-126 in myocardial tissue of rat was decreased in the early stage of severe burn injury. It may participate in regulating myocardial damage and play a protective role.

Animals ; Burns ; metabolism ; pathology ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Hypoxia ; MicroRNAs ; genetics ; metabolism ; Myocardium ; metabolism ; pathology ; Myocytes, Cardiac ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Serum ; Soft Tissue Injuries ; Transfection ; Troponin I ; metabolism