Multimodal ischemic stroke recurrence prediction model based on the capsule neural network and support vector machine.

Ischemic stroke (IS) has a high recurrence rate. Machine learning (ML) models have been developed based on single-modal biochemical tests, and imaging data have been used to predict stroke recurrence. However, the prediction accuracy of these models is not sufficiently high. Therefore, this study aimed to collect biochemical detection and magnetic resonance imaging (MRI) data to establish a dataset and propose a high-performance heterogeneous multimodal IS recurrence prediction model based on deep learning. This is a retrospective cohort study. Data were retrospectively collected from 634 IS patients in Zhuhai, China, a 12-month follow-up was conducted to determine stroke recurrence. We propose the ischemic stroke multi-group learning (ISGL) model, an integrated model for predicting the recurrence risk of multimodal IS in patients, based on a capsule neural network and a linear support vector machine (SVM). Two capsule neural network prediction models based on T1 and T2 signals in the MRI data and a SVM prediction model based on biochemical test data were established. Finally, a vote was conducted on the final judgment of the integrated model. The ISGL model was compared with 6 classical ML and deep learning models: k-nearest neighbors, SVM, logistic regression, random forest, eXtreme Gradient Boosting, and visual geometry group. The results revealed that the accuracy, specificity, sensitivity and the area under the curve of the ISGL model were 95%, 96%, 94%, and 95%, respectively. Among the comparison models, the visual geometry group method exhibited the best performance, but it much lower than those of the ISGL model. Analysis of the importance of biochemical test data revealed that low-density lipoprotein, smoking, and heart disease history were the positively correlated factors, and total cholesterol, high-density lipoprotein, and diabetes were and the negatively correlated factors. This study proposes the ISGL model can be used simultaneously with MRI and biochemical data to predict IS recurrence. This combination resulted in higher rate of performance than that of the other ML models. Additionally, this study found related risk factors affected recurrence, which can be used to intervene in high-risk patients' recurrence as early as possible and promote the development of secondary prevention of stroke.

Total tanshinones ameliorates cGAS-STING-mediated inflammatory and autoimmune diseases by affecting STING-IRF3 binding.

BACKGROUND: An important signaling pathway connecting illness and natural immunity is the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, but aberrant activation of this pathway is associated with the development of autoimmune and inflammatory diseases. Hence, targeted inhibition of the activation of the cGAS-STING pathway is potentially valuable in the treatment of disease. The primary active component of Salvia miltiorrhiza is total tanshinone (TTN). Research has indicated that TTN possesses noteworthy anti-inflammatory properties. However, the protective mechanism of TTN against acute liver injury (ALI) and autoimmune diseases is unknown. METHODS: A model of aberrant activation of the cGAS-STING pathway was established in various cells and treated with TTN, and the expression of cGAS-STING pathway-related proteins, type I interferon, interferon stimulated genes and inflammatory factors was assessed by western blotting, real-time qPCR. Immunofluorescence analysis of the effect of TTN on the entry of associated proteins into the nucleus following aberrant activation of the cGAS-STING pathway. The effect of TTN on STING oligomerisation was investigated using 2'-3'-cyclic GMP-AMP (2',3'-cGAMP) to induce STING oligomerisation. Western blotting was used to examine the impact of TTN on the interactions of STING, tank-binding kinase 1 (TBK1), and interferon regulatory factor 3 (IRF3) after HA or Flag-labelled plasmids were transfected into HEK-293 T cells. A dimethylxanthenone-4-acetic acid (DMXAA) -induced activation model of the cGAS-STING pathway in mice was established to study the effect of TTN on aberrant activation of the cGAS-STING pathway in vivo. On the other hand, an animal model of lipopolysaccharide/D-galactosamine (LPS/D-GaIN)-induced ALI and an autoimmune disease model induced by trex1 knockout were established to study the effects of TTN on inflammatory and autoimmune diseases mediated by the cGAS-STING pathway in vivo. RESULTS: In several models of aberrant activation of the cGAS-STING pathway, TTN significantly inhibited the phosphorylation of STING and IRF3, thereby suppressing the expression of type I interferon, interferon-stimulated genes and inflammatory factors. Additionally, TTN prevented P65 and IRF3 from entering the nucleus after the cGAS-STING signalling pathway was abnormally activated. Subsequent research indicated that TTN was not involved in the oligomerization of STING or the integration of STING-TBK1 and TBK1-IRF3. However, TTN was found to have a substantial effect on the binding process between STING and IRF3. On the other hand, DMXAA-induced STING activation and activation of downstream signalling in vivo are inhibited by TTN. Furthermore, TTN exhibits positive treatment effects on autoimmune diseases caused by deficiency of trex1 and LPS/D-GaIN-induced ALI. CONCLUSION: Our research indicates that TTN effectively treats ALI and autoimmune illnesses mediated by the cGAS-STING pathway by inhibiting the abnormal activation of this pathway.

Psychometric testing of the training needs for advance care planning scale for clinicians and nurses.

BACKGROUND: Studies have shown that Chinese Clinicians and nurses have positive attitudes toward ACP, but no local tools exist to assess their need for ACP knowledge and skills training. resulting in their inability to initiate ACP conversations as well as poor end-of-life care for patients. Therefore, this study aims to assess the needs of Chinese Clinicians and nurses for ACP knowledge and skills training and assess the validity and reliability of a questionnaire on the Training Needs for Advance Care Planning (TNACP) scale. METHODS: From October to November 2021, 170 clinicians and nurses were pre-surveyed using a preliminary draft of the questionnaire. The responses were screened using item analysis, Cronbach's alpha coefficient, and the intraclass correlation coefficient (ICC) to describe the internal consistency and stability of the questionnaire. The Content validity index (CVI), Exploratory factor analysis (EFA) and Confirmatory factor analysis (CFA) were used to test the validity of the questionnaire. RESULTS: After independent samples t-test analysis, Except for the entry "A2", the critical ratio between the two groups of the remaining 23 items was statistically significant (p < 0.05). Based on the above screening methods, the "A2" item was deleted, and the final number of questionnaire items was 23. The I-CVI was 0.79-1.00, and the S-CVI/Ave was 0.90. Three common factors were extracted-the cumulative contribution rate was 69.969%, and the factor loading of all items was 0.506-0.843 (all > 0.40). The results of confirmatory factor analysis showed that the Training Needs for Advance Care Planning (TNACP) scale model fit well(X2/df = 2.504, RMSEA = 0.092, GFI = 0.809, AGFI = 0.745, CFI = 0.931, IFI = 0.932, TLI = 0.916); the Cronbach's α = 0.888 for the total questionnaire, and the three dimensions of Cronbach's α were 0.729 to 0.959; and the ICC for the overall scores between the test-retest evaluations was 0.884 (p < 0.001). CONCLUSIONS: The TNACP scale has good reliability and validity and can be used to assess Chinese Clinicians and nurses' training needs for implementing ACP.

An Integrated Multi-omics prediction model for stroke recurrence based on Lnet transformer layer and dynamic weighting mechanism.

BACKGROUND AND OBJECTIVE: Stroke is a disease with high mortality and disability. Importantly, the fatality rate demonstrates a significant increase among patients afflicted by recurrent strokes compared to those experiencing their initial stroke episode. Currently, the existing research encounters three primary challenges. The first is the lack of a reliable, multi-omics image dataset related to stroke recurrence. The second is how to establish a high-performance feature extraction model and eliminate noise from continuous magnetic resonance imaging (MRI) data. The third is how to integration multi-omics data and dynamically weighted for different omics data. METHODS: We systematically compiled MRI and conventional detection data from a cohort comprising 737 stroke patients and established PSTSZC, a multi-omics dataset for predicting stroke recurrence. We introduced the first-ever Integrated Multi-omics Prediction Model for Stroke Recurrence, MPSR, which is based on ResNet, Lnet-transformer, LSTM and dynamically weighted DNN. The MPSR model comprises two principal modules, the Feature Extraction Module, and the Integrated Multi-Omics Prediction Module. In the Feature Extraction module, we proposed a novel Lnet regularization layer, which effectively addresses noise issues in MRI data. In the Integrated Multi-omics Prediction Module, we propose a dynamic weighted mechanism based on evaluators, which mitigates the noise impact brought about by low-performance omics. RESULTS: We compared seven single-omics models and six state-of-the-art multi-omics stroke recurrence models. The experimental results demonstrate that the MPSR model exhibited superior performance. The accuracy, AUROC, specificity, and sensitivity of the MPSR model can reach 0.96, 0.97, 1, and 0.94, respectively, which is higher than the results of contrast model. CONCLUSION: MPSR is the first available high-performance multi-omics prediction model for stroke recurrence. We assert that the MPSR model holds the potential to function as a valuable tool in assisting clinicians in accurately diagnosing individuals with a predisposition to stroke recurrence.

Experiences and perspectives of healthcare professionals, patients and caregivers toward the serious illness conversation guide: protocol for a qualitative meta-synthesis.

INTRODUCTION: Exploring the experiences and perspectives of healthcare professionals, patients and caregivers toward serious illness conversations based on the Serious Illness Care Program is vital for improving communication with patients who are seriously ill, as has been shown in previous studies. However, few studies have carried out a systematic review to examine common themes, strengthen conclusions and identify gaps in the literature, the findings of which could help steer further research, policies and practice to improve more timely and person-centred conversations about the values and priorities of patients with serious illnesses. The objective of this qualitative meta-synthesis is to explore how healthcare professionals, patients and caregivers described their own experiences of the processes of serious illness communication through a secondary analysis of published qualitative data. METHODS AND ANALYSIS: Meta-aggregation will be used to conduct a systematic review of qualitative studies. We conducted an initial search on 10 October 2023; papers published in English will be searched using electronic databases, including PubMed (MEDLINE), Web of Science, Embase, Ovid and CINAHL. Studies that satisfy the eligibility criteria will be evaluated for methodological quality using the Joanna Briggs Institute (JBI) Critical Appraisal Checklist for Qualitative Research. The meta-aggregative review will consist of the following: (1) extraction of findings of all included studies; (2) categorisation of the findings, with at least two findings per category; and (3) synthesis of one or more findings from at least two categories. Study eligibility screening, data extraction, analysis and JBI Critical Appraisal Checklist and Grading of Recommendations Assessment, Development and Evaluation-Confidence in the Evidence from Reviews of Qualitative research assessments will be undertaken independently by two authors. ETHICS AND DISSEMINATION: Secondary data analysis of published literature does not require ethical approval. The results will be disseminated in peer-reviewed journals and presented in conference papers and elsewhere. PROSPERO REGISTRATION NUMBER: CRD42022330859.

Applications of Machine Learning to Diagnosis of Parkinson's Disease.

BACKGROUND: Accurate diagnosis of Parkinson's disease (PD) is challenging due to its diverse manifestations. Machine learning (ML) algorithms can improve diagnostic precision, but their generalizability across medical centers in China is underexplored. OBJECTIVE: To assess the accuracy of an ML algorithm for PD diagnosis, trained and tested on data from different medical centers in China. METHODS: A total of 1656 participants were included, with 1028 from Beijing (training set) and 628 from Fuzhou (external validation set). Models were trained using the least absolute shrinkage and selection operator-logistic regression (LASSO-LR), decision tree (DT), random forest (RF), eXtreme gradient boosting (XGboost), support vector machine (SVM), and k-nearest neighbor (KNN) techniques. Hyperparameters were optimized using five-fold cross-validation and grid search techniques. Model performance was evaluated using the area under the curve (AUC) of the receiver operating characteristic (ROC) curve, accuracy, sensitivity (recall), specificity, precision, and F1 score. Variable importance was assessed for all models. RESULTS: SVM demonstrated the best differentiation between healthy controls (HCs) and PD patients (AUC: 0.928, 95% CI: 0.908-0.947; accuracy: 0.844, 95% CI: 0.814-0.871; sensitivity: 0.826, 95% CI: 0.786-0.866; specificity: 0.861, 95% CI: 0.820-0.898; precision: 0.849, 95% CI: 0.807-0.891; F1 score: 0.837, 95% CI: 0.803-0.868) in the validation set. Constipation, olfactory decline, and daytime somnolence significantly influenced predictability. CONCLUSION: We identified multiple pivotal variables and SVM as a precise and clinician-friendly ML algorithm for prediction of PD in Chinese patients.

Experiences and perspectives of healthcare professionals implementing advance care planning for people suffering from life-limiting illness: a systematic review and meta-synthesis of qualitative studies.

BACKGROUND: Life-limited patients may lose decision-making abilities during disease progression. Advance care planning can be used as a discussion method for healthcare professionals to understand patients' future care preferences. However, due to many difficulties, the participation rate of healthcare professionals in advance care planning is not high. AIM: To explore the facilitators of and barriers to healthcare professionals' provision of advance care planning to life-limited patients to better implement it for this population. METHODS: We followed ENTREQ and PRISMA to guide this study. We conducted a systematic search of PubMed, Web of Science, Embase, CINAHL, PsycINFO, CNKI, and SinoMed to include qualitative data on the experiences and perspectives of healthcare professionals in different professional fields in providing advance care planning for life-limited patients. The Joanna Briggs Institute Critical Appraisal Checklist for Qualitative Research was used to assess the quality of the included studies. RESULTS: A total of 11 studies were included. Two themes were identified: unsupported conditions and facilitative actions. Healthcare professionals regarded cultural concepts, limited time, and fragmented record services as obstacles to implementation. They had low confidence and were overly concerned about negative effects. They needed to possess multiple abilities, learn to flexibly initiate topics, and facilitate effective communication based on multidisciplinary collaboration. CONCLUSION: Healthcare professionals need an accepting cultural environment to implement advance care planning, a sound legal system, financial support, and a coordinated and shared system to support them. Healthcare systems need to develop educational training programs to increase the knowledge and skills of healthcare professionals and to promote multidisciplinary collaboration to facilitate effective communication. Future research should compare the differences in the needs of healthcare professionals in different cultures when implementing advance care planning to develop systematic implementation guidelines in different cultures.

Ursolic acid inhibits human dermal fibroblasts hyperproliferation, migration, and collagen deposition induced by TGF-ß via regulating the Smad2/3 pathway.

Hypertrophic scar (HS) is a skin condition characterized by excessive fibrosis with disordered collagens from skin fibroblasts, which causes abnormal esthetic and even functional symptoms, thereby affecting millions of people. Ursolic acid (UA) is widely used in skincare and exerts anti-fibrotic effects. The present study aimed to delve into the impact of UA on HS and the mechanism. Fibroblasts (FBs) were incubated with TGF-ß to investigate physiological characteristics compared with FBs isolated from normal skin (NSFBs) and hyperplastic scars (HSFBs). TGF-ß-incubated FBs were subjected to treatment with UA (0-20 µM). The expressions of Vimentin, α-SMA, Collagen I, and Collagen III were examined using immunofluorescence, RT-qPCR, and western blot. Cell viability, proliferation, apoptosis, migration, and contractility were examined by CCK-8, EdU, Annexin V-FITC/PI, Transwell, and collagen gel contraction assays, respectively. The activation of Smad2/3 signaling was also determined by western blot. The binding sites for UA of TGF-ßR1 (ALK5) were predicted by the Autodock tool. Compared with NSFBs, the cell proliferation, migration, and contractility of both HSFBs and TGF-ß-incubated FBs were all significantly up-regulated. UA markedly impaired the TGF-ß-induced increase in cell proliferation, migration, and contractility, α-SMA, collagen I, and Collagen III expression of FBs. UA significantly inhibited the phosphorylation levels of Smad2/3 in TGF-ß-incubated FBs with no influence on TGF-ßR1 and TGF-ßR2 expressions, which might be because of the binding of UA to the catalytic domain of ALK5 protein. UA attenuated TGF-ß1-induced hyperproliferation, migration, and collagen deposition in FBs via regulating the Smad2/3 pathway.

Integrated transgene and transcriptome reveal the molecular basis of MdWRKY87 positively regulate adventitious rooting in apple rootstock.

For most fruit and forest species vegetative propagated from elite genotypes, adventitious rooting is essential. The ability to form adventitious roots significantly decreased during the juvenile to adult phase change. Apart from the miR156-SPL pathway, whether there is another regulation mechanism controlling age-dependent adventitious rooting ability remained largely unknown. In the present study, we showed that MdWRKY87 expression level was positively correlation with adventitious rooting ability. In addition, over-expressing of MdWRKY87 in tobacco leads to enhanced adventitious rooting ability, more adventitious root number and accelerated adventitious rooting process. Comparative transcriptome profiling indicated that MdWRKY87 overexpression can activate the expression of adventitious rooting-induced genes, such as WOX11 and AIL. In addition, MdWRKY87 overexpression can inhibit the transcription of adventitious rooting-repressed genes, such as AUX/IAAs and type-B cytokinin RRs. Collectively, here we demonstrated that higher expression level of MdWRKY87 contributes to age-dependent adventitious rooting-competent in juvenile apple rootstock.

Efficient degradation of ivermectin by newly isolated Aeromonas taiwanensis ZJB-18,044.

Ivermectin (IVM) is a widely used antiparasitic agent and acaricide. Despite its high efficiency against nematodes and arthropods, IVM may pose a threat to the environment due to its ecotoxcity. In this study, degradation of IVM by a newly isolated bacterium Aeromonas taiwanensis ZJB-18,044 was investigated. Strain ZJB-18,044 can completely degrade 50 mg/L IVM in 5 d with a biodegradation ability of 0.42 mg/L/h. Meanwhile, it exhibited high tolerance (50 mg/L) to doramectin, emamectin, rifampicin, and spiramycin. It can also efficiently degrade doramectin, emamectin, and spiramycin. The IVM degradation of strain ZJB-18,044 can be inhibited by erythromycin, azithromycin, spiramycin or rifampicin. However, supplement of carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation, can partially recover the IVM degradation. Moreover, strain ZJB-18,044 cells can pump out excess IVM to maintain a low intracellular IVM concentration. Therefore, the IVM tolerance of strain ZJB-18,044 may be due to the regulation of the intracellular IVM concentration by the activated macrolide efflux pump(s). With the high IVM degradation efficiency, A. taiwanensis ZJB-18,044 may serve as a bioremediation agent for IVM and other macrolides in the environment.

Enhanced (R)-2-(4-Hydroxyphenoxy)Propionic Acid Production by Beauveria bassiana: Optimization of Culture Medium and H2O2 Supplement Under Static Cultivation.

(R)-2-(4-hydroxyphenoxy)propionic acid (HPOPA) is a key intermediate for the preparation of aryloxyphenoxypropionic acid herbicides (R-isomer). In order to improve the HPOPA production from the substrate (R)-2-phenoxypropionic acid (POPA) with Beauveria bassiana CCN-A7, static cultivation and H2O2 addition were attempted and found to be conducive to the task at hand. This is the first report on HPOPA production under static cultivation and reactive oxygen species (ROS) induction. On this premise, the cultivation conditions and fermentation medium compositions were optimized. As a result, the optimal carbon source, organic nitrogen source, and inorganic nitrogen source were determined to be glucose, peptone, and ammonium sulfate, respectively. The optimal inoculum size and fermentation temperature were 13.3% and 28°C, respectively. The significant factors including glucose, peptone, and H2O2, identified based on Plackett-Burman design, were further optimized through Central Composite Design (CCD). The optimal concentrations/amounts were as follows: glucose 38.81 g/l, peptone 7.28 g/l, and H2O2 1.08 ml/100 ml. Under the optimized conditions, HPOPA titer was improved from 9.60 g/l to 19.53 g/l, representing an increase of 2.03- fold. The results obtained in this work will provide novel strategies for improving the biosynthesis of hydroxy aromatics.

Production of (R)-2-(4-hydroxyphenoxy) propionic acid by Beauveria bassiana ZJB16007 in solid state fermentation using rice bran.

R-2-(4-hydroxyphenoxy)propionic acid (R-HPPA) is a key chiral intermediate for phenoxypropionic acid herbicide synthesis. In this study, to improve the production of R-HPPA with B. bassiana ZJB16007, the cultivation conditions in solid-state fermentation (SSF) were investigated. The effects of various substrates on R-HPPA production were evaluated and the process parameters were also optimized. The results showed that rice bran was the optimal substrate for R-HPPA production. The optimal medium components and cultivation conditions were: rice bran: silkworm chrysalis powder = 5.25: 2.25 (g: g), nutrient salts solution 12 mL which contained 50 g/L R-PPA, pH 5.0, and cultivated at 28 °C for 11 days. Under the optimized conditions, the transformation of R-HPPA was significantly improved and the yield of R-HPPA reached 77.78%, which was 15.14% higher than that of the control (67.55%). Therefore, SSF may serve as an alternative for R-HPPA production by B. bassiana ZJB16007.

Genome-Wide Association Study Identifies Candidate Genes Related to Seed Oil Composition and Protein Content in Gossypium hirsutum L.

Cotton (Gossypium spp.) is a leading natural fiber crop and an important source of vegetable protein and oil for humans and livestock. To investigate the genetic architecture of seed nutrients in upland cotton, a genome-wide association study (GWAS) was conducted in a panel of 196 germplasm resources under three environments using a CottonSNP80K chip of 77,774 loci. Relatively high genetic diversity (average gene diversity being 0.331) and phenotypic variation (coefficient of variation, CV, exceeding 3.9%) were detected in this panel. Correlation analysis revealed that the well-documented negative association between seed protein (PR) and oil may be to some extent attributable to the negative correlation between oleic acid (OA) and PR. Linkage disequilibrium (LD) was unevenly distributed among chromosomes and subgenomes. It ranged from 0.10-0.20 Mb (Chr19) to 5.65-5.75 Mb (Chr25) among the chromosomes and the range of Dt-subgenomes LD decay distances was smaller than At-subgenomes. This panel was divided into two subpopulations based on the information of 41,815 polymorphic single-nucleotide polymorphism (SNP) markers. The mixed linear model considering both Q-matrix and K-matrix [MLM(Q+K)] was employed to estimate the association between the SNP markers and the seed nutrients, considering the false positives caused by population structure and the kinship. A total of 47 SNP markers and 28 candidate quantitative trait loci (QTLs) regions were found to be significantly associated with seven cottonseed nutrients, including protein, total fatty acid, and five main fatty acid compositions. In addition, the candidate genes in these regions were analyzed, which included three genes, Gh_D12G1161, Gh_D12G1162, and Gh_D12G1165 that were most likely involved in the control of cottonseed protein concentration. These results improved our understanding of the genetic control of cottonseed nutrients and provided potential molecular tools to develop cultivars with high protein and improved fatty acid compositions in cotton breeding programs through marker-assisted selection.

Genome-wide analysis of codon usage bias in four sequenced cotton species.

Codon usage bias (CUB) is an important evolutionary feature in a genome which provides important information for studying organism evolution, gene function and exogenous gene expression. The CUB and its shaping factors in the nuclear genomes of four sequenced cotton species, G. arboreum (A2), G. raimondii (D5), G. hirsutum (AD1) and G. barbadense (AD2) were analyzed in the present study. The effective number of codons (ENC) analysis showed the CUB was weak in these four species and the four subgenomes of the two tetraploids. Codon composition analysis revealed these four species preferred to use pyrimidine-rich codons more frequently than purine-rich codons. Correlation analysis indicated that the base content at the third position of codons affect the degree of codon preference. PR2-bias plot and ENC-plot analyses revealed that the CUB patterns in these genomes and subgenomes were influenced by combined effects of translational selection, directional mutation and other factors. The translational selection (P2) analysis results, together with the non-significant correlation between GC12 and GC3, further revealed that translational selection played the dominant role over mutation pressure in the codon usage bias. Through relative synonymous codon usage (RSCU) analysis, we detected 25 high frequency codons preferred to end with T or A, and 31 low frequency codons inclined to end with C or G in these four species and four subgenomes. Finally, 19 to 26 optimal codons with 19 common ones were determined for each species and subgenomes, which preferred to end with A or T. We concluded that the codon usage bias was weak and the translation selection was the main shaping factor in nuclear genes of these four cotton genomes and four subgenomes.

[Effects of recombinant human granulocyte-macrophage colony-stimulating factor on wound healing and microRNA expression in diabetic rats].

OBJECTIVE: To investigate the effects of recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) on wound healing and microRNA expression in diabetic rats. METHODS: Eighteen male SD rats of clean grade were used to reproduce diabetes model. Four weeks later, a total of 64 full-thickness skin wounds were created on the back of 16 rats with established diabetes, with 4 wounds on each rat. Two symmetrical wounds on either side of the spine were created as a pair according to paired design. Then the wounds were divided into groups A and B according to the random number table and blind method (red and blue tags on the rhGM-CSF or the gel vehicle), with 32 wounds in each group. The ointment with red tag was applied on the wounds of group A and the blue one on group B. The application was conducted once a day, with a thickness of 3 mm, up to post injury day (PID) 14. Gross observation of wound healing was conducted on PID 3, 7, 14. The wound healing rate was determined on PID 3 and 7. On PID 3, 7, 14, tissues from 2, 4, and 8 wounds were harvested from each group respectively for the observation of the histopathological changes with HE staining, and also for analyzing the expression of proliferating cell nuclear antigen (PCNA) and CD31 with immunohistochemical staining (denoted as absorbance value). On PID 7, tissues from 6 wounds in each group were harvested for microarray gene chip to screen the differentially expressed microRNAs. Enrichment analysis of Kyoto encyclopedia of genes and genomes (KEGG) signaling pathway on the differentially expressed microRNAs were performed after the microRNA screening results were validated by real-time fluorescent quantitative RT-PCR. Data were processed with paired t test or two-sample t test. RESULTS: (1) On PID 3, the wound area was significantly decreased, and the wound granulation was significantly proliferated in both groups. On PID 7, the wound area was further decreased, and the wound area was almost filled by granulation in both groups; the conditions in group A were better. On PID 14, all the wounds in group A were almost healed, while a small area of raw wound with incrustation still remained in some wounds of group B. On PID 3 and 7, the wound healing rates of group A were (41 ± 5)% and (75 ± 4)%, significantly higher than those of group B [(31 ± 9)% and (71 ± 4)%, with t values respectively 10.13 and 8.06, P values below 0.001]. (2) On PID 3, the epidermal cells, endothelial cells, and Fbs in the wounds of 2 groups were sparse, with heavy infiltration of inflammatory cells. The above condition in the wounds was better in group A than in group B. On PID 7, the epidermal cells, endothelial cells, and Fbs were gradually well arranged in group A; infiltration of inflammatory cells decreased, and the condition was better than that of group B. On PID 14, the wounds of group A were completely covered by epidermis, while infiltration of inflammatory cells still remained in some wounds of group B. (3) On PID 3, 7, 14, the positive expressions of CD31 and PCNA in group A were respectively 0.275 ± 0.018, 0.345 ± 0.034, 0.305 ± 0.023; 0.406 ± 0.063, 0.223 ± 0.011, 0.045 ± 0.022. They were significantly higher than those of group B (0.222 ± 0.020, 0.229 ± 0.018, 0.197 ± 0.015; 0.324 ± 0.039, 0.162 ± 0.012, 0.018 ± 0.020, with t values from 2.281 to 9.652, P < 0.05 or P < 0.01). (4) According to the microRNAs detection and screening, as compared with group B, 18 microRNAs were up-regulated while 13 were down-regulated in the wounds of group A. (5) The results of real-time fluorescent quantitative RT-PCR had good consistency with the results of microRNAs detection. (6) Enrichment analysis of KEGG signaling pathway showed that among the 31 differentially expressed microRNAs, 4 took part in the MAPK signaling pathway, 3 took part in the Wnt signaling pathway, 1 took part in the TGF-ß signaling pathway, 3 took part in the epidermal growth factor receptor signaling pathway, 2 took part in the cell cycle pathway, 5 took part in the axon guidance signaling pathway, 6 took part in the focal adhesion pathway, 3 took part in the regulation of actin cytoskeleton pathway, 1 took part in the extracellular cell matrix receptor pathway, 3 took part in the adherens junction pathway, and 1 took part in the cell adhesion molecules pathway. After disclosing the blind, it showed that the ointment with red tag was the rhGM-CSF gel and the blue one was gel vehicle. CONCLUSIONS: The rhGM-CSF gel can promote wound healing in diabetic rats, producing significant differential microRNA expression in wounds, and they may be the target at gene post-transcriptional level of rhGM-CSF gel in promoting wound healing.