MicroRNA-544 inhibits inflammatory response and cell apoptosis after cerebral ischemia reperfusion by targeting IRAK4.

OBJECTIVE: Stroke remains the most common malignant cerebrovascular event in the world. The correlation between the expression of miR-544 and the degree of cerebral ischemia reperfusion (CIR) injury has not been well recognized in recent years. This study focuses on the effect of miR-544 on inflammation and apoptosis after CIR. PATIENTS AND METHODS: Plasma expression of miR-544 in ischemic stroke (IS) patients and healthy controls was determined by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The effects of miR-544 on cerebral infarction and neurological deficits were verified in vitro by tail vein injection of Ago-miR-544. Western blotting was utilized to examine protein expressions of key proteins involving in inflammation and apoptosis in mouse brain. Western blotting, immunofluorescence staining and luciferase assays were used to demonstrate whether miR-544 influences the expression of interleukin-1 receptor-associated kinase 4 (IRAK4), downstream inflammatory and apoptosis-related proteins. RESULTS: MiR-544 was found decreased in peripheral blood of IS patients compared with healthy controls. MiR-544 has been shown to relieve neurological deficits and reduce the volume of cerebral infarction in mice. Overexpression of miR-544 ameliorated the inflammation and apoptotic responses in brain tissue after ischemia reperfusion by down-regulating the expression of IRAK4, whereas the low expression was opposite in vivo and in vitro. CONCLUSIONS: We found that miR-544 may participate in controlling inflammation and apoptosis after ischemia-reperfusion by targeting IRAK4, providing possible diagnostic indicators and therapeutic targets for IS.

Correlations of neuronal apoptosis with expressions of c-Fos and c-Jun in rats with post-ischemic reconditioning damage.

OBJECTIVE: Transcription factors (c-Fos and c-Jun) have been considered to play roles in the initiation of programmed nerve cell death. However, the roles of c-Fos and c-Jun protein expressions in neuronal apoptosis of rats with post-ischemic reconditioning damage were not clarified. Therefore, the aim of this study was to investigate the correlations of protein expressions of c-Fos and c-Jun with neuronal apoptosis of rats with post-ischemic reconditioning damage. MATERIALS AND METHODS: Rat models of post-ischemic reconditioning were established firstly. Then, apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay, and the gene expression levels of apoptosis-related proteins [cytochrome c (Cyt c), B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax)] were detected by reverse transcription-polymerase chain reaction (RT-PCR). Lastly, Western blotting was used to determine the protein expression levels of c-Fos and c-Jun, and the expressions of c-Fos and c-Jun in brain tissues of models were measured by immunohistochemistry. RESULTS: Treatment group had significantly increased malonaldehyde (MDA) level and significantly decreased superoxide dismutase (SOD) activity in rat cortex compared with those in control group (p<0.05). The number of TUNEL positive cells in the right cortex of rats in the treatment group was clearly higher than that in control group. Among them, post-ischemic reperfusion group had reduced level of Bax in the cytoplasm, but increased Bax level in the mitochondrion, and lowered expression level of Bcl-2 in both mitochondrion and cytoplasm in comparison with control group. Dynamic detection results of c-Jun were in synchronization with those of apoptosis proteins, and maximum expression occurred at 24 h after treatment. CONCLUSIONS: c-Jun may play a role in the initiation of apoptotic cell death in these neurons.

Research on the nutrition and cognition of high-risk stroke groups in community and the relevant factors.

OBJECTIVE: To investigate the prevalence rate of nutritional risk in high-risk stroke groups in community, analyze its influencing factors, and analyze and compare the relationship between nutritional risk or malnutrition assessed by different nutritional evaluation methods and cognitive function, so as to provide the basis and guidance for clinical nutritional assessment and support. PATIENTS AND METHODS: A cross-sectional survey was performed for 1196 cases in high-risk stroke groups in community from December 2015 to January 2017. At the same time, the nutritional status of patients was evaluated using the mini nutritional assessment (MNA) and MNA-short form (MNA-SF), and the cognitive status of patients was evaluated using the mini-mental state examination (MMSE). Moreover, the relevant influencing factors of nutritional risk and MMSE score were analyzed and compared. RESULTS: High-risk stroke groups in community suffered from a high risk of malnutrition. MNA-SF had a higher specificity and lower false positive rate than MNA. Nutritional risk occurred more easily in high-risk stroke groups in community with a history of diabetes mellitus, less physical exercise or light manual labor, daily use of multiple drugs, and higher age. Those with a higher nutritional risk were more prone to cognitive impairment. High-risk stroke groups in community, complicated with hyperhomocysteinemia, daily use of three or more kinds of prescription drugs, and a previous history of stroke, were accompanied by cognitive impairment easily. CONCLUSIONS: MNA-SF can be used for the nutritional screening of high-risk stroke groups in community. For the high-risk stroke groups in community, the rational nutritional diet should be publicized, blood sugar should be controlled in a scientific manner and physical exercise should be moderately increased.

[Clinicopathologic features of adenoid cystic carcinoma of breast].

Objective: To investigate the clinicopathologic features and grading of adenoid cystic carcinoma (ACC) of the breast. Methods: Sixteen cases of ACC of the breast were analyzed and graded according to the previous report. Immunohistochemical (IHC) staining was used to detect the immunophenotype, Ki-67 proliferative index and expression of EZH2, and the association with tumor grade and outcome was analyzed. Results: Of the 16 cases, 11 were grade Ⅰ, with the epithelial and myoepithelial cells being arranged into tubular and cribriform structure with no solid component; three were grade Ⅱ, which were composed of mixed tubular, cribriform and solid component (<30%); and two were grade Ⅲ, which showed mainly solid component (>90%) and the tumor cells showed basaloid features with scanty cytoplasm and hyperchromatic nuclei, and mitotic count was>5/10 HPF. Immunophenotypically, the epithelial cells expressed CK7, CK8/18 and CD117; the myoepithelial cells expressed p63 and CK5/6; while the basaloid cells were positive for CK5/6 and CD117.Tubular and cibriform ACC showed low Ki-67 and EZH2 expression, while the two cases of solid variant with basaloid features showed high level of Ki-67 and EZH2 expression. Follow-up data were available in 13 cases with a median follow-up period of 42 months. Lung metastasis occurred after 12 months in one grade Ⅱ case and the patient died of disease after 34 months. Vertebral metastasis occurred after 12 months in one grade Ⅲ case and axillary lymph node metastasis occurred in another grade Ⅲ case. All other patients were free of disease at the end of the follow-up periods. Conclusions: ACC shows morphologic spectrum varying from low to high grade, the latter can may give rise to local and distant metastasis. ACC should not be regarded simply as low malignant potential, and should be graded for optimal treatment.

[Effects of human amniotic epithelial stem cells-derived exosomes on healing of wound with full-thickness skin defect in rats].

Objective: To investigate the effects of human amniotic epithelial stem cells-derived exosomes on healing of wound with full-thickness skin defect in rats. Methods: (1) Human amniotic epithelial stem cells were isolated from the amnion tissue of 5 full-term pregnant women in Department of Obstetrics of our hospital by the method of trypsin digestion, and their morphology was observed. The third passage of cells were stained with rhodamine-phalloidin for cytoskeleton observation. The third passage of cells were identified with flow cytometry through the detection of expressions of cell surface markers CD29, CD31, CD34, CD90, CD105, SSEA3, SSEA4 and immunity-related marker human leukocyte antigen-D related site (HLA-DR). The third passage of cells were also assessed the ability of adipogenic and osteogenic differentiation. (2) The third passage of human amniotic epithelial stem cells were cultured in DMEM medium supplemented with 10% exosome-free fetal bovine serum. Exosomes were isolated from culture supernatant by the method of ultracentrifugation and represented with scanning electron microscope for morphologic observation. (3) Six adult SD rats were anesthetized, and four 1 cm×1 cm sized wounds with full-thickness skin defect were made on the back of each rat. The wounds on the back of each rat were divided into control group, 25 µg/mL exosomes group, 50 µg/mL exosomes group, and 100 µg/mL exosomes group according to the random number table (with 6 wounds in each group), and a total volume of 100 µL phosphate buffered saline, 25 µg/mL exosomes, 50 µg/mL exosomes, and 100 µg/mL exosomes were evenly injected around the wound through multiple subcutaneous sites, respectively. The wound healing rate was calculated based on measurement on post injury day (PID) 7, 14, and 21. On PID 21, the healed wound tissue of each group was collected and stained with HE to observe and count skin accessories, and the arrangement of collagen fibers was observed with Masson staining. Data were processed with analysis of variance for repeated measurement, analysis of variance of randomized block design, one-way analysis of variance, and Bonferroni test. Results: (1) The cells, which were isolated and cultured, displayed typical cobblestone morphology with many microvilli on cell surface. Among the cells, the positive expression rates of CD29, CD90, SSEA3, and SSEA4 were above 50.0%, and the rate of CD105 was 8.0%, while the rates of CD31, CD34, and HLA-DR were almost 0. The cells could differentiate into adipocytes and osteoblasts. The above results revealed that the cells cultured were human amniotic epithelial stem cells. (2) Human amniotic epithelial stem cells-derived exosomes were round or oval vesicles with diameter from 50 to 150 nm. (3) On PID 7 and 21, wound healing rates of the four groups were close (with P values above 0.05). On PID 14, wound healing rates of 50 and 100 µg/mL exosomes groups were (89.8±4.3)% and (92.0±4.6)% respectively, significantly higher than the wound healing rate of control group [(80.3±6.4)%, P<0.05 or P<0.01]. Moreover, the wound healing rate of 100 µg/mL exosomes group was significantly higher than that of 25 µg/mL exosomes group [(83.3±5.1)%, P<0.05]. On PID 21, the numbers of skin accessories in 50 and 100 µg/mL exosomes groups were 4.3±1.4 and 5.1±1.6 respectively, obviously more than those of control group and 25 µg/mL exosomes group (respectively 1.4±0.5 and 1.8±0.6, with P values below 0.01). Well reorganized collagen fibers were observed just in the healed wound tissue of 50 and 100 µg/mL exosomes groups. Conclusions: Human amniotic epithelial stem cells-derived exosomes can promote healing of wound with full-thickness skin defect in rats.