This study aimed to explore the mechanisms and molecular targets of total flavones of Abelmoschus manihot(TFA) plus empagliflozin(EM) in attenuating diabetic tubulopathy(DT) by targeting mitochondrial homeostasis and pyroptosis-apoptosis-necroptosis(PANoptosis). In the in vivo study, the authors established the DT rat models through a combination of uninephrectomy, administration of streptozotocin via intraperitoneal injections, and exposure to a high-fat diet. Following modeling successfully, the DT rat models received either TFA, EM, TFA+EM, or saline(as a vehicle) by gavage for eight weeks, respectively. In the in vitro study, the authors subjected the NRK52E cells with or without knock-down Z-DNA binding protein 1(ZBP1) to a high-glucose(HG) environment and various treatments including TFA, EM, and TFA+EM. In the in vivo and in vitro studies, The authors investigated the relative characteristics of renal tubular injury and renal tubular epithelial cells damage induced by reactive oxygen species(ROS), analyzed the relative characteristics of renal tubular PANoptosis and ZBP1-mediatted PANoptosis in renal tubular epithelial cells, and compared the relative characteristics of the protein expression levels of marked molecules of mitochondrial fission in the kidneys and mitochondrial homeostasis in renal tubular epithelial cells, respectively. Furthermore, in the network pharmacology study, the authors predicted and screened targets of TFA and EM using HERB and SwissTargetPrediction databases; The screened chemical constituents and targets of TFA and EM were constructed the relative network using Cytoscape 3.7.2 network graphics software; The relative targets of DT were integrated using OMIM and GeneCards databases; The intersecting targets of TFA, EM, and DT were enriched and analyzed signaling pathways by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG) software using DAVID database. In vivo study results showed that TFA+EM could improve renal tubular injury, the protein expression levels and characteristics of key signaling molecules in PANoptosis pathway in the kidneys, and the protein expression levels of marked molecules of mitochondrial fission in the kidneys. And that, the ameliorative effects in vivo of TFA+EM were both superior to TFA or EM. Network pharmacology study results showed that TFA+EM treated DT by regulating the PANoptosis signaling pathway. In vitro study results showed that TFA+EM could improve ROS-induced cell injury, ZBP1-mediatted PANoptosis, and mitochondrial homeostasis in renal tubular epithelial cells under a state of HG, including the protein expression levels of marked molecules of mitochondrial fission, mitochondrial ultrastructure, and membrane potential level. And that, the ameliorative effects in vitro of TFA+EM were both superior to TFA or EM. More importantly, using the NRK52E cells with knock-down ZBP1, the authors found that, indeed, ZBP1 was mediated PANoptosis in renal tubular epithelial cells as an upstream factor. In addition, TFA+EM could regulate the protein expression levels of marked signaling molecules of PANoptosis by targeting ZBP1. In summary, this study clarified that TFA+EM, different from TFA or EM, could attenuate DT with multiple targets by ameliorating mitochondrial homeostasis and inhibiting ZBP1-mediated PANoptosis. These findings provide the clear pharmacological evidence for the clinical treatment of DT with a novel strategy of TFA+EM, which is named "coordinated traditional Chinese and western medicine".
Animals ; Rats ; Mitochondria/metabolism* ; Benzhydryl Compounds/administration & dosage* ; Glucosides/administration & dosage* ; Abelmoschus/chemistry* ; Male ; Homeostasis/drug effects* ; Flavones/administration & dosage* ; Rats, Sprague-Dawley ; Diabetic Nephropathies/physiopathology* ; Drugs, Chinese Herbal/administration & dosage* ; DNA-Binding Proteins/genetics* ; Humans ; Apoptosis/drug effects*

It has become an industry consensus that self-assembled nanoparticles (SAN) are formed by molecular recognition of chemical components in traditional Chinese medicine during the decoction process. The insoluble components in the decoction are mostly in the form of nanoparticles, which can improve the problem of poor water solubility. However, the transfer rate of these insoluble components in the decoction is still very low, which limits the efficacy of the drug. This study aimed to refine the traditional decoction self-assembly phenomenon. The self-assembled nanoparticles were constructed by micro-precipitation method (MP-SAN), and characterized by particle size, zeta potential, stability index and morphology. The formation of MP-SAN and alterations in related physicochemical properties were evaluated using modern spectroscopic and thermal analysis techniques. The quality value transmitting pattern of lignan components within the MP-SAN was assessed via high performance liquid chromatography (HPLC). The MP-SAN showed sphere-like structure with uniform morphology, particle size of (245.3 ± 3.2) nm, polydispersity index (PDI) of (0.13 ± 0.03), zeta potential of (-48.9 ± 5.9) mV and stability index (SI) of (86.05% ± 2.27%). Comprehensive analyses using ultraviolet visible spectroscopy, Fourier transform infrared spectroscopy, differential scanning calorimetry, and other techniques confirmed molecular recognition between the decoction and ethanol extraction, leading to electron rearrangement under the influence of non-covalent bonding. This resulted in the formation of nanoparticles possessing superior thermal stability. As determined by HPLC, the encapsulation rates of the index components in the MP-SAN were all greater than 75% (dehydrodiconiferyl alcohol: 77.00%; herpetolide A: 78.57%; herpetrione: 94.53%), and the transfer rates were all higher than 65% (dehydrodiconiferyl alcohol: 96.01%; herpetolide A: 67.86%; herpetrione: 65.55%), which were 1.34, 1.38 and 4.81 times compared with those of the traditional decoction. In summary, this study successfully constructed the MP-SAN based on micro-precipitation method to achieve high transfer rate and high encapsulation rate of insoluble components in docoction, which provides a pharmaceutics idea for the efficient utilization of pharmacodynamic substance basis of traditional Chinese medicine.

Objective:To investigate the effect of GRIM-19 on the resistance of carcinoma cells to the chemotherapeutic agent docetaxel in the treatment of PCa.Methods:Using siRNA technology to interfere with the gene expression in PCa cells,we estab-lished a model of GRIM-19 overexpression/knockdown in PCa cells.We investigated the effect of different expression levels of GRIM-19 on docetaxel-induced death of the PCa cells by qPCR,Western blot and flow cytometry,and assessed the value of GRIM-19 in re-ducing the chemotherapy-resistance of PCa cells.Results:GRIM-19 was down-regulated in PCa tissues and cells.Knockout of GRIM-19 significantly decreased the expression of siGRIM19 in the PC-3 and LNCaP cells,and reduced their death rate when treated with docetaxel compared with the control group.The expressions of GRIM-19 mRNA and protein were remarkably upregulated after transfection with GRIM-19,and the overexpressed GRIM-19 promoted the death of the PC-3 and LNCaP cells treated with docetaxel in a dose-dependent manner.Flow cytometry analysis showed a lower apoptosis rate of PC-3-R cells than that of PC-3 cells at different time points of docetaxel-induction at different doses.Conclusion:GRIM-19 is a PCa suppressor gene with a significant facilitating effect on the apoptosis of PCa cells,and the overexpression of GRIM-19 promotes docetaxel-induced PCa cell death and improves the sensitivity of chemotherapy.

Pulmonary blast injury has become the main type of trauma in modern warfare, characterized by externally mild injuries but internally severe injuries, rapid disease progression, and a high rate of early death. The injury is complicated in clinical practice, often with multiple and compound injuries. Currently, there is a lack of effective protective materials, accurate injury detection instrument and portable monitoring and transportation equipment, standardized clinical treatment guidelines in various medical centers, and evidence-based guidelines at home and abroad, resulting in a high mortality in clinlcal practice. Therefore, the Trauma Branch of Chinese Medical Association and the Editorial Committee of Chinese Journal of Trauma organized military and civilian experts in related fields such as thoracic surgery and traumatic surgery to jointly develop the Clinical treatment guideline for pulmonary blast injury ( version 2023) by combining evidence for effectiveness and clinical first-line treatment experience. This guideline provided 16 recommended opinions surrounding definition, characteristics, pre-hospital diagnosis and treatment, and in-hospital treatment of pulmonary blast injury, hoping to provide a basis for the clinical treatment in hospitals at different levels.

Objective: To construct and verify a light-weighted convolutional neural network (CNN), and explore its application value for screening the early stage (subcategory 0/1 and stage Ⅰ of pneumoconiosis) of coal workers' pneumoconiosis (CWP) from digital chest radiography (DR) . Methods: A total of 1225 DR images of coal workers who were examined at an Occupational Disease Prevention and Control Institute in Anhui Province from October 2018 to March 2021 were retrospectively collected. All DR images were collectively diagnosed by 3 radiologists with diagnostic qualifications and gave diagnostic results. There were 692 DR images with small opacity profusion 0/- or 0/0 and 533 DR images with small opacity profusion 0/1 to stage Ⅲ of pneumoconiosis. The original chest radiographs were preprocessed differently to generate four datasets, namely 16-bit grayscale original image set (Origin16), 8-bit grayscale original image set (Origin 8), 16-bit grayscale histogram equalized image set (HE16) and 8-bit grayscale histogram equalized image set (HE8). The light-weighted CNN, ShuffleNet, was applied to train the generated prediction model on the four datasets separately. The performance of the four models for pneumoconiosis prediction was evaluated on a test set containing 130 DR images using measures such as the receiver operating characteristic (ROC) curve, accuracy, sensitivity, specificity, and Youden index. The Kappa consistency test was used to compare the agreement between the model predictions and the physician diagnosed pneumoconiosis results. Results: Origin16 model achieved the highest ROC area under the curve (AUC=0.958), accuracy (92.3%), specificity (92.9%), and Youden index (0.8452) for predicting pneumoconiosis, with a sensitivity of 91.7%. And the highest consistency between identification and physician diagnosis was observed for Origin16 model (Kappa value was 0.845, 95%CI: 0.753-0.937, P<0.001). HE16 model had the highest sensitivity (98.3%) . Conclusion: The light-weighted CNN ShuffleNet model can efficiently identify the early stages of CWP, and its application in the early screening of CWP can effectively improve physicians' work efficiency.
Humans ; Retrospective Studies ; Anthracosis/diagnostic imaging* ; Pneumoconiosis/diagnostic imaging* ; Coal Mining ; Neural Networks, Computer ; Coal

Objective: Compare and analyze the results of the domestic Lanyi AH600 glycated hemoglobin analyzer and other different detection systems to understand the comparability of the detection results of different detectors, and establish the best cut point of Lanyi AH600 determination of haemoglobin A1c (HbA1c) in the diagnosis of diabetes. Methods: Multi center cohort study was adopted. The clinical laboratory departments of 18 medical institutions independently collected test samples from their respective hospitals from March to April 2022, and independently completed comparative analysis of the evaluated instrument (Lanyi AH600) and the reference instrument HbA1c. The reference instruments include four different brands of glycosylated hemoglobin meters, including Arkray, Bio-Rad, DOSOH, and Huizhong. Scatter plot was used to calculate the correlation between the results of different detection systems, and the regression equation was calculated. The consistency analysis between the results of different detection systems was evaluated by Bland Altman method. Consistency judgment principles: (1) When the 95% limits of agreement (95% LoA) of the measurement difference was within 0.4% HbA1c and the measurement score was≥80 points, the comparison consistency was good; (2) When the measurement difference of 95% LoA exceeded 0.4% HbA1c, and the measurement score was≥80 points, the comparison consistency was relatively good; (3) The measurement score was less than 80 points, the comparison consistency was poor. The difference between the results of different detection systems was tested by paired sample T test or Wilcoxon paired sign rank sum test; The best cut-off point of diabetes was analyzed by receiver operating characteristic curve (ROC). Results: The correlation coefficient R² of results between Lanyi AH600 and the reference instrument in 16 hospitals is≥0.99; The Bland Altman consistency analysis showed that the difference of 95% LoA in Nanjing Maternity and Child Health Care Hospital in Jiangsu Province (reference instrument: Arkray HA8180) was -0.486%-0.325%, and the measurement score was 94.6 points (473/500); The difference of 95% LoA in the Tibetan Traditional Medical Hospital of TAR (reference instrument: Bio-Rad Variant II) was -0.727%-0.612%, and the measurement score was 89.8 points; The difference of 95% LoA in the People's Hospital of Chongqing Liang Jiang New Area (reference instrument: Huizhong MQ-2000PT) was -0.231%-0.461%, and the measurement score was 96.6 points; The difference of 95% LoA in the Taihe Hospital of traditional Chinese Medicine in Anhui Province (reference instrument: Huizhong MQ-2000PT) was -0.469%-0.479%, and the measurement score was 91.9 points. The other 14 hospitals, Lanyi AH600, were compared with 4 reference instrument brands, the difference of 95% LoA was less than 0.4% HbA1c, and the scores were all greater than 95 points. The results of paired sample T test or Wilcoxon paired sign rank sum test showed that there was no statistically significant difference between Lanyi AH600 and the reference instrument Arkray HA8180 (Z=1.665,P=0.096), with no statistical difference. The mean difference between the measured values of the two instruments was 0.004%. The comparison data of Lanyi AH600 and the reference instrument of all other institutions had significant differences (all P<0.001), however, it was necessary to consider whether it was within the clinical acceptable range in combination with the results of the Bland-Altman consistency analysis. The ROC curve of HbA1c detected by Lanyi AH600 in 985 patients with diabetes and 3 423 patients with non-diabetes was analyzed, the area under curve (AUC) was 0.877, the standard error was 0.007, and the 95% confidence interval 95%CI was (0.864, 0.891), which was statistically significant (P<0.001). The maximum value of Youden index was 0.634, and the corresponding HbA1c cut point was 6.235%. The sensitivity and specificity of diabetes diagnosis were 76.2% and 87.2%, respectively. Conclusion: Among the hospitals and instruments currently included in this study, among these four hospitals included Nanjing Maternity and Child Health Care Hospital in Jiangsu Province (reference instrument: Arkray HA8180), Tibetan Traditional Medical Hospital of TAR (reference instrument: Bio-Rad Variant Ⅱ), the People's Hospital of Chongqing Liang Jiang New Area (reference instrument: Huizhong MQ-2000PT), and the Taihe Hospital of traditional Chinese Medicine in Anhui Province (reference instrument: Huizhong MQ-2000PT), the comparison between Lanyi AH600 and the reference instruments showed relatively good consistency, while the other 14 hospitals involved four different brands of reference instruments: Arkray, Bio-Rad, DOSOH, and Huizhong, Lanyi AH600 had good consistency with its comparison. The best cut point of the domestic Lanyi AH600 for detecting HbA1c in the diagnosis of diabetes is 6.235%.
Pregnancy ; Child ; Humans ; Female ; Glycated Hemoglobin ; Cohort Studies ; Diabetes Mellitus/diagnosis* ; Sensitivity and Specificity ; ROC Curve

OBJECTIVE: To establish a model of long-term free drinking mouse by feeding mice with alcohol to simulate the state of human voluntary long-term drinking, and on this basis, to further discuss the evaluation criteria of long-term free drinking mice model in sports, anxiety and cognitive behavior. METHODS: Forty six-week-old SPF C57BL/6 male mouse were randomly divided into two groups: Long-term free drinking group (n=20) and normal control group (n=20). The two groups were given solid feed normally. The long-term free drinking group was free to take 10% alcohol and water every day, while the normal drinking group only took water every day. The mice were fed for 7 months, and were evaluated by a series of behavioral methods, including Rota-rod test, balance beam test, open filed test, the elevated plus maze, two-box social behavior, new object recognition, Y maze and water maze. RESULTS: With the increase of drinking days, the mice showed significant alcohol addiction in the alcohol preference test. With the increase of alcohol intake, the mice in the long-term free choice drinking group had slightly shiny fur and reduced diet. Compared with the control group, the weight gain began to slow down from the third month, and the weight decreased significantly by the sixth and seventh months (P=0.006, P < 0.001). The mice showed reduced balance locomotion ability (P=0.003, P=0.001) in the rotary bar and balance beam test. In the open field and elevated cross test, the mice had obvious anxiety-like behavior (P < 0.001). The mice showed decreased social ability in the two boxes of social behavior (P < 0.016). In the experiment of new object recognition and Y maze, the exploration of new object decreased (P=0.018, P=0.040). In the water maze, cognitive functions, such as learning and spatial memory were reduced (P < 0.001). CONCLUSION The successful establishment of the long-term free drinking mouse model is more convenient for us to carry out further research on the neural mechanism of alcohol addiction, and lays an experimental foundation for exploring the neural mechanism of alcohol addiction and related new targets.
Mice ; Male ; Humans ; Animals ; Alcoholism ; Mice, Inbred C57BL ; Alcohol Drinking/psychology* ; Anxiety ; Disease Models, Animal ; Ethanol

OBJECTIVES: To study the biological processes and functions of serum exosomes in children in the acute stage of Kawasaki disease (KD), so as to provide new biomarkers for the early diagnosis of KD. METHODS: In this prospective study, 13 children with KD who were treated in Children's Hospital of Soochow University from June 2019 to August 2020 were enrolled as the KD group, and 13 children who were hospitalized due to bacterial infection during the same period were enrolled as the control group. Whole blood was collected on the next morning after admission, serum samples were obtained by centrifugation, and exosomes were extracted through ultracentrifugation. Serum exosomes were analyzed by label-free quantitative proteomics, and differentially expressed proteins (DEPs) were screened out for functional enrichment analysis. A protein-protein interaction (PPI) network was plotted, and unique proteins were validated by targeted proteomics. RESULTS: A total of 131 DEPs were screened out for the two groups, among which 27 proteins were detected in both groups. There were 48 unique DEPs in the KD group, among which 23 were upregulated and 25 were downregulated, and these proteins acted on "complement and coagulation cascades" and "the MAPK signaling pathway". Validation by targeted proteomics showed that FGG, SERPING1, C1R, C1QA, IGHG4, and C1QC proteins were quantifiable in the KD group. A total of 29 proteins were only expressed in the control group, among which 12 were upregulated and 17 were downregulated. Four proteins were quantifiable based on targeted proteomics, i.e., VWF, ECM1, F13A1, and TTR. A PPI network was plotted for each group. In the KD group, FGG and C1QC had close interaction with other proteins, while in the control group, VWF had close interaction with other proteins. CONCLUSIONS The serum exosomes FGG and C1QC in children in the acute stage of KD are expected to become the biomarkers for the early diagnosis of KD. For children with unexplained fever, detection of FGG, C1QC1, and VWF may help with etiological screening.
Biomarkers ; Child ; Exosomes ; Extracellular Matrix Proteins ; Humans ; Mucocutaneous Lymph Node Syndrome/diagnosis* ; Prospective Studies ; Proteomics ; von Willebrand Factor

OBJECTIVE: To observe the inhibitory effect of Shenbai Jiedu Fang (SBJDF, a compound recipe of traditional Chinese herbal drugs) on chemically induced carcinogenesis of colorectal adenoma in mice and explore the role of PTEN/PI3K/AKT signaling pathway in mediating this effect. METHODS: Four-week-old male C57BL/6 mice were randomly divided into control group (n=10), AOM/DSS model group (n=20), low-dose (14 g/kg) SBJDF group (n=10) and high-dose (42 g/kg) SBJDF group (n= 10). In the latter 3 groups, the mice were treated with azoxymethane (AOM) and dextran sodium sulphate (DSS) to induce carcinogenesis of colorectal adenoma. In the two SBJDF treatment groups, SBJDF was administered daily by gavage during the modeling. The survival rate, body weight, general condition of the mice, and intestinal adenoma formation and carcinogenesis were observed. The expressions of proteins associated with the PTEN/PI3K/AKT signaling pathway in the intestinal tissue were detected using immunohistochemistry. RESULTS: Compared with those in the model group, the mice treated with SBJDF, especially at the high dose, showed a significantly lower incidence of intestinal carcinogenesis and had fewer intestinal tumors with smaller tumor volume. Pathological examination showed the occurrence of adenocarcinoma in the model group, while only low-grade and high-grade neoplasia were found in low-dose SBJDF group; the mice treated with high-dose SBJDF showed mainly normal mucosal tissues in the intestines with only a few lesions of low-grade neoplasia of adenoma. Compared with those in the control group, the mice in the model group had significantly elevated plasma miRNA-222 level (P < 0.05), which was obviously lowered in the two SBJDF groups (P < 0.01). The results of immunohistochemistry revealed that compared with the model group, the two SBJDF groups, especially the high-dose group, had significantly up-regulated expressions of PTEN, P-PTEN and GSK-3β and down-regulated expressions of p-GSK-3 β, PI3K, AKT, P-AKT, β-catenin, c-myc, cyclinD1 and survivin in the intestinal tissues. CONCLUSION SBJDF can significantly inhibit colorectal adenoma formation and carcino-genesis in mice possibly through regulating miRNA-222 and affecting PTEN/PI3K/AKT signaling pathway.
Animals ; Male ; Mice ; Adenoma/prevention & control* ; Azoxymethane/adverse effects* ; Carcinogenesis/drug effects* ; Colorectal Neoplasms/prevention & control* ; Dextran Sulfate/adverse effects* ; Disease Models, Animal ; Glycogen Synthase Kinase 3 beta/metabolism* ; Mice, Inbred C57BL ; MicroRNAs/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Proto-Oncogene Proteins c-akt/metabolism* ; Signal Transduction ; Drugs, Chinese Herbal/therapeutic use*

Objective: To investigate the effects of exosomes from human adipose-derived mesenchymal stem cells (ADSCs) on inflammatory response of mouse RAW264.7 cells and wound healing of full-thickness skin defects in mice. Methods: The experimental research methods were adopted. The discarded adipose tissue was collected from 3 female patients (aged 10-25 years) who underwent abdominal surgery in the First Affiliated Hospital of Air Force Medical University. ADSCs were extracted from the adipose tissue by collagenase Ⅰ digestion and identified with flow cytometry. Exosomes were extracted from the human ADSCs by differential ultracentrifugation, the morphology of the exosomes was observed by transmission electron microscopy, the particle diameter of the exosomes was detected by nanoparticle tracking analyzer, and the protein expressions of CD9, CD63, tumor susceptibility gene 101 (TSG101), and β-actin were detected by Western blotting. The human ADSCs exosomes (ADSCs-Exos) and RAW264.7 cells were co-cultured for 12 h, and the uptake of RAW264.7 cells for human ADSCs-Exos was observed. The RAW264.7 cells were divided into phosphate buffer solution (PBS) group stimulated with PBS for suitable time, endotoxin/lipopolysaccharide (LPS) stimulation 2 h group, LPS stimulation 4 h group, LPS stimulation 6 h group, LPS stimulation 12 h group, and LPS stimulation 24 h group stimulated with LPS for corresponding time, with 3 wells in each group, and the mRNA expressions of interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), IL-6, and IL-10 were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR) method. The RAW264.7 cells were divided into PBS group, LPS alone group, and LPS+ADSCs-Exos group, with 3 wells in each group, which were dealt correspondingly for the time screened out in the previous experiment, the mRNA expressions of IL-1β, TNF-α, IL-6, IL-10, trasforming growth factor β (TGF-β,) and vascular endothelial growth factor (VEGF) were detected by real time fluorescence quantitative RT-PCR method, and the protein expressions of inducible nitric oxide synthase (iNOS) and arginase 1 (Arg1) were detected by Western blotting. Twenty-four 8-week-old male BALB/c mice were divided into PBS group and ADSCs-Exos group according to the random number table, with 12 mice in each group, and a full-thickness skin defect wound with area of 1 cm×1 cm was inflicted on the back of each mouse. Immediately after injury, the wounds of mice in the two groups were dealt correspondingly. On post injury day (PID) 1, the concentration of IL-1β and TNF-α in serum were detected by enzyme-linked immunosorbent assay, and the mRNA expressions of IL-1β, TNF-α, and IL-6 were detected by real time fluorescence quantitative RT-PCR method. On PID 3, 6, 9, 12, and 15, the wound healing was observed and the wound non-healing rate was calculated. On PID 15, the defect length of skin accessory and collagen volume fraction (CVF) were detected by hematoxylin eosin staining and Masson staining, respectively, the CD31 expression and neovascularization were detected by immunohistochemistry, and the ratio of Ki67 positive cells, the ratio of iNOS and Arg1 double positive cells, and the ratio of iNOS positive cells to Arg1 positive cells and their fluorescence intensities were detected by immunofluorescence method. The number of samples in animal experiments was 6. Data were statistically analyzed with analysis of variance for repeated measurement, one-way analysis of variance, and independent sample t test. Results: At 12 h of culture, the cells exhibited a typical spindle shape, which were verified as ADSCs with flow cytometry. The exosomes with a vesicular structure and particle diameters of 29-178 nm, were positively expressed CD9, CD63, and TSG101 and negatively expressed β-actin. After 12 h of co-culture, the human ADSCs-Exos were endocytosed into the cytoplasm by RAW264.7 cells. The mRNA expressions of IL-1β, TNF-α, IL-6, and IL-10 of RAW264.7 cells in LPS stimulation 2 h group, LPS stimulation 4 h group, LPS stimulation 6 h group, LPS stimulation 12 h group, and LPS stimulation 24 h group were significantly higher than those in PBS group (with t) values of 39.10, 14.55, 28.80, 4.74, 48.80, 22.97, 13.25, 36.34, 23.12, 18.71, 29.19, 41.08, 11.68, 18.06, 8.54, 43.45, 62.31, 22.52, 21.51, and 37.13, respectively, P<0.01). The stimulation 12 h with significant expressions of all the inflammatory factors was selected as the time point in the following experiment. After stimulation of 12 h, the mRNA expressions of IL-1β, TNF-α, IL-6, and IL-10 of RAW264.7 cells in LPS alone group were significantly higher than those in PBS group (with t values of 44.20, 51.26, 14.71, and 8.54, respectively, P<0.01); the mRNA expressions of IL-1β, TNF-α, and IL-6 of RAW264.7 cells in LPS+ADSCs-Exos group were significantly lower than those in LPS alone group (with t values of 22.89, 25.51, and 8.03, respectively, P<0.01), while the mRNA expressions of IL-10, TGF-β, and VEGF were significantly higher than those in LPS alone group (with t values of 9.89, 13.12, and 7.14, respectively, P<0.01). After stimulation of 12 h, the protein expression of iNOS of RAW264.7 cells in LPS alone group was significantly higher than that in PBS group and LPS+ADSCs-Exos group, respectively (with t values of 11.20 and 5.06, respectively, P<0.05 or P<0.01), and the protein expression of Arg1 was significantly lower than that in LPS+ADSCs-Exos group (t=15.01, P<0.01). On PID 1, the serum concentrations of IL-1β and TNF-α and the mRNA expressions of IL-1β, TNF-α, and IL-6 in wound tissue of mice in ADSCs-Exos group were significantly those in lower than PBS group (with t values of 15.44, 12.24, 9.24, 7.12, and 10.62, respectively, P<0.01). On PID 3, 6, 9, 12, and 15 d, the wound non-healing rates of mice in ADSCs-Exos group were (73.2±4.1)%, (53.8±3.8)%, (42.1±5.1)%, (24.1±2.8)%, and 0, which were significantly lower than (82.5±3.8)%, (71.2±4.6)%, (52.9±4.1)%, (41.5±3.6)%, and (14.8±2.5)% in PBS group, respectively (with t values of 4.77, 8.93, 5.54, 7.63, and 7.59, respectively, P<0.01). On PID 15, the defect length of skin accessory in wounds of mice in PBS group was significantly longer than that in ADSCs-Exos group (t=9.50, P<0.01), and the CVF was significantly lower than that in ADSCs-Exos group (t=9.15, P<0.01). On PID 15, the CD31 expression and the number of new blood vessels (t=12.99, P<0.01), in wound tissue of mice in ADSCs-Exos group were significantly more than those in PBS group, and the ratio of Ki67 positive cells was significantly higher than that in PBS group (t=7.52, P<0.01). On PID 15, the ratio of iNOS and Arg1 double positive cells in wound tissue of mice in PBS group was (12.33±1.97)%, which was significantly higher than (1.78±0.29)% in ADSCs-Exos group (t=13.04, P<0.01), the ratio of iNOS positive cells and the fluorescence intensity of iNOS were obviously higher than those of ADSCs-Exos group, and the ratio of Arg1 positive cells and the fluorescence intensity of Arg1 were obviously lower than those of ADSCs-Exos group. Conclusions: The human ADSCs-Exos can alleviate inflammatory response of mouse RAW264.7 cells, decrease macrophage infiltration and secretion of the pro-inflammatory cytokines, increase the secretion of anti-inflammatory cytokines to promote neovascularization and cell proliferation in full-thickness skin defect wounds of mice, hence accelerating wound healing.
Animals ; Exosomes ; Female ; Humans ; Male ; Mesenchymal Stem Cells ; Mice ; Skin ; Vascular Endothelial Growth Factor A ; Wound Healing