Objective:To investigate whether 6-shogaol (6-SH) alleviates oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal autophagy and calcium overload by promoting the expression of microRNA-26a-5p (miR-26a-5p) and inhibiting death-associated protein kinase 1 (DAPK1), and to explore its potential mechanisms.Methods:Primary cultured logarithmic growth phase mouse hippocampal neurons HT22 cells were taken and cell counting kit-8 (CCK-8) was used to detect cell viability, searching for the optimal concentration of Na 2S 2O 4. HT22 cells were divided into blank control group (NC group), OGD/R group (sugar-free culture medium + 10 mmol/L Na 2S 2O 4 treatment for 1.5 hours followed by normal culture medium for 4 hours), 6-SH intervention group (cultured with 10 μmol/L 6-SH for 4 hours after OGD), negative control inhibitor pretreatment group (transfected with negative control inhibitor for 48 hours followed by OGD, then cultured with 6-SH for 4 hours), and miR-26a-5p inhibitor pretreatment group (transfected with miR-26a-5p inhibitor for 48 hours followed by OGD, then cultured with 6-SH for 4 hours). Cell viability of each group was detected by CCK-8 method; cell ultrastructure was observed under transmission electron microscopy; real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the gene expressions of DAPK1 and miR-26a-5p; molecular docking were used to verify the interaction between 6-SH and miR-26a-5p; dual-luciferase assay was used to verify the targeting relationship between DAPK1 and miR-26a-5p; flow cytometry was used to determine the levels of intracellular Ca 2+; Western blotting was used to detect the protein expressions of phosphorylated-glutamate receptor 2B (p-NMDAR2B) Ser1303, DAPK1, autophagy related protein Beclin1, light chain 3 (LC3), and p-DAPK1 Ser308; immunofluorescence was used to detect the expression of LC3 and Beclin1. Results:The results of the CCK-8 assay showed that the cell viability of the 6-SH intervention group was significantly increased compared to the OGD/R group, while the cell viability of the miR-26a-5p inhibitor pretreatment group was significantly decreased compared to the 6-SH intervention group. Transmission electron microscopy revealed that the number of autophagosomes in the 6-SH intervention group was significantly reduced compared to the OGD/R group, while the number of autophagosomes in the miR-26a-5p inhibitor pretreatment group was significantly increased compared to the 6-SH intervention group. RT-qPCR results showed that compared with the OGD/R group, the expression of miR-26a-5p was significantly upregulated and the expression of DAPK1 mRNA was significantly downregulated in the 6-SH intervention group; compared with the 6-SH intervention group, the expression of miR-26a-5p was significantly downregulated and the expression of DAPK1 mRNA was significantly upregulated in the miR-26a-5p inhibitor pretreatment group. Molecular docking verified the interaction between 6-SH and miR-26a-5p. Dual-luciferase reporter gene assay showed that compared with the negative control group, mmu-miR-26a-5p significantly downregulated the luciferase expression of m-DAPK1-3UTR-WT, indicating a binding interaction between them. Flow cytometry results showed that compared with the OGD/R group, the level of intracellular Ca 2+ was significantly decreased in the 6-SH intervention group; compared with the 6-SH intervention group, the level of Ca 2+ was significantly increased in the miR-26a-5p inhibitor pretreatment group. Western blotting results showed that compared with the OGD/R group, the protein expressions of p-NMDAR2B Ser1303, DAPK1, Beclin1, and LC3 were significantly decreased in the 6-SH intervention group (p-NMDAR2B Ser1303/β-actin: 2.34±0.27 vs. 4.78±0.39, DAPK1/β-actin: 1.40±0.13 vs. 2.37±0.21, Beclin1/β-actin: 2.61±0.32 vs. 4.32±0.29, LC3/β-actin: 2.52±0.45 vs. 5.09±0.18, all P < 0.05), while the protein expression of p-DAPK1 Ser308 was significantly increased (p-DAPK1 Ser308/β-actin: 0.66±0.09 vs. 0.40±0.02, P < 0.05); compared with the 6-SH intervention group, the protein expressions of p-NMDAR2B Ser1303, DAPK1, Beclin1, and LC3 were significantly increased in the miR-26a-5p inhibitor pretreatment group (p-NMDAR2B Ser1303/β-actin: 4.08±0.14 vs. 2.34±0.27, DAPK1/β-actin: 1.96±0.15 vs. 1.40±0.13, Beclin1/β-actin: 3.92±0.31 vs. 2.61±0.32, LC3/β-actin: 4.33±0.33 vs. 2.52±0.45, all P < 0.05), while the expression of p-DAPK1 Ser308 protein was significantly decreased (p-DAPK1 Ser308/β-actin: 0.33±0.12 vs. 0.66±0.09, P < 0.05); immunofluorescence staining showed that compared with the OGD/R group, the fluorescence intensity of LC3 and Beclin1 was significantly decreased in the 6-SH intervention group; compared with the 6-SH intervention group, the fluorescence intensity of LC3 and Beclin1 was significantly increased in the miR-26a-5p inhibitor pretreatment group. Conclusion:6-SH can alleviate neuronal damage by regulating miR-26a-5p/DAPK1 to reduce autophagy and calcium overload in cells.

OBJECTIVE: To investigate whether hydrogen-rich water exerts a protective effect against cellular injury by affecting the level of autophagy after oxygen glucose deprivation/reoxygenation (OGD/R) in a mouse hippocampal neuronal cell line (HT22 cells). METHODS: HT22 cells in logarithmic growth phase were cultured in vitro. Cell viability was detected by cell counting kit-8 (CCK-8) assay to find the optimal concentration of Na₂S₂O₄. HT22 cells were divided into control group (NC group), OGD/R group (sugar-free medium+10 mmol/L Na₂S₂O₄ treated for 90 minutes and then changed to normal medium for 4 hours) and hydrogen-rich water treatment group (HW group, sugar-free medium+10 mmol/L Na₂S₂O₄ treated for 90 minutes and then changed to medium containing hydrogen-rich water for 4 hours). The morphology of HT22 cells was observed by inverted microscopy; cell activity was detected by CCK-8 method; cell ultrastructure was observed by transmission electron microscopy; the expression of microtubule-associated protein 1 light chain 3 (LC3) and Beclin-1 was detected by immunofluorescence; the protein expression of LC3II/I and Beclin-1, markers of cellular autophagy, was detected by Western blotting. RESULTS: Inverted microscopy showed that compared with the NC group, the OGD/R group had poor cell status, swollen cytosol, visible cell lysis fragments and significantly lower cell activity [(49.1±2.7)% vs. (100.0±9.7)%, P < 0.01]; compared with the OGD/R group, the HW group had improved cell status and remarkably higher cell activity [(63.3±1.8)% vs. (49.1±2.7)%, P < 0.01]. Transmission electron microscopy showed that the neuronal nuclear membrane of cells in the OGD/R group was lysed and a higher number of autophagic lysosomes were visible compared with the NC group; compared with the OGD/R group, the neuronal damage of cells in the HW group was reduced and the number of autophagic lysosomes was notably decreased. The results of immunofluorescence assay showed that the expressions of LC3 and Beclin-1 were outstandingly enhanced in the OGD/R group compared with the NC group, and the expressions of LC3 and Beclin-1 were markedly weakened in the HW group compared with the OGD/R group. Western blotting assay showed that the expressions were prominently higher in both LC3II/I and Beclin-1 in the OGD/R group compared with the NC group (LC3II/I: 1.44±0.05 vs. 0.37±0.03, Beclin-1/β-actin: 1.00±0.02 vs. 0.64±0.01, both P < 0.01); compared with the OGD/R group, the protein expression of both LC3II/I and Beclin-1 in the HW group cells were notably lower (LC3II/I: 0.54±0.02 vs. 1.44±0.05, Beclin-1/β-actin: 0.83±0.07 vs. 1.00±0.02, both P < 0.01). CONCLUSIONS Hydrogen-rich water has a significant protective effect on OGD/R-causing HT22 cell injury, and the mechanism may be related to the inhibition of autophagy.
Mice ; Animals ; Oxygen/metabolism* ; Beclin-1/pharmacology* ; Glucose/metabolism* ; Actins ; Sincalide ; Autophagy/physiology* ; Hydrogen/pharmacology* ; Reperfusion Injury ; Apoptosis

Objective:To construct a targeted and accurate evaluation system for facial and cervical wounds and scars of burn patients.Methods:The method combining literature analysis and survey research was adopted, and the basic principles of item system construction were followed. From June to August 2020, based on the aesthetic standards of facial and cervical plastic surgery, the topographic map assessment system for facial and cervical wounds and scars of burn patients was preliminarily formed, focusing on the assessment of wounds and scars in the necks and faces of patients after burns. In September 2020, 38 experts in the relevant fields were consulted in advance and the questionnaire was revised according to the experts' opinions. From December 2020 to March 2021, the Delphi method was applied to conduct inquiry by correspondence with 35 experts in relevant fields from Guangzhou, Shenzhen, Shanghai, Beijing, and other cities, who met the inclusion criteria, and the items were screened and established. The effective recovery rate of inquiry questionnaire was calculated to determine the level of enthusiasm of experts, the average authority coefficient of all items was calculated to determine the level of expert authority, the average importance expert score, the average coefficient of variation, and the average full score rate of all the third-level items were calculated to determine the concentration of expert opinions, the average coefficients of variation and Kendall's harmony coefficients of the importance, sensitivity, and operability expert scores of all the third-level items were calculated to determine the degree of coordination of expert opinions. The Kendall's harmony coefficients for the importance, sensitivity, and operability expert scores of all the third-level items were statistically analyzed with chi-square test.Results:Among the 35 experts consulted by Delphi method, mainly were male, aged (48±10) years, with 8-38 years of working experience, mainly with associate senior titles and above, all with a bachelor's degree or above education background, and of whom 11 were burn experts, 7 were wound repair experts, 4 were plastic surgery experts, and 13 were rehabilitation medicine experts. Finally, a topographic map assessment system for facial and cervical wounds and scars of burn patients was formed, including 4 first-level items, 21 second-level items, 40 third-level items, and 1 mask. The effective recovery rate of inquiry questionnaire was 100% (35/35). The average authority coefficient of all items was 0.89. The average importance expert score was 4.67, the average coefficient of variation of importance expert score was 0.01, and the average full score rate of all the third-level items was 86.3%. The average coefficients of variation of the importance, sensitivity, and operability expert scores of all the third-level items were 0.01, 0.01, and 0.02, respectively. The Kendall's harmony coefficients for the importance, sensitivity, and operability expert scores of all the third-level items were statistically significant (with χ2 values of 1 201.53, 745.67, and 707.07, respectively , P<0.05). Conclusions:The established topographic map assessment system for facial and cervical wounds and scars of burn patients has high scientificity and reliability, which can be used for the evaluation of facial and neck wounds or scars in burn patients.

Objective:To evaluate the safety and short-term outcomes of transfemoral transcatheter aortic valve replacement (TAVR) with domestic prostheses in patients with pure native aortic valve regurgitation (AR).Methods:A total of 16 patients with pure native AR who underwent transfemoral TAVR in the Renmin Hospital of Wuhan University from June 2019 to January 2022 were consecutively included in our study, and 24 patients with aortic stenosis (AS) who underwent transfemoral TAVR in the same period were selected as the control group. This study compared the baseline characteristics, baseline echocardiography, morphological characteristics of the aortic root, safety of the procedure and short-term outcomes between the two groups.Results:Compared with the AS group, the pure native AR group had a higher prevalence of baseline NYHA class Ⅲ or Ⅳ, a larger left ventricular end-diastolic diameter (LVEDD), a smaller relative ventricular wall thickness (RWT) (all P<0.05), a lower aortic root calcification score, and a larger sinus junction diameter, and cardiac angle (all P<0.05). During TAVR operation, the pure native AR group was treated with larger prostheses size, with a larger percentage in relation to the native annulus size and outflow tract (all P<0.05). There were 7 cases (43.8%) treated with 'valve in valve’, 2 cases (12.5%) with moderate paravalvular leak(PVL), and 2 cases (12.5%) with prostheses-migration to ascending aorta.However, no cases of death, transfer to surgery, coronary obstruction or annular rupture were observed in the pure native AR group. There were no statistical differences between the pure native AR group and AS group in device success rate (56.3% vs 62.5%, P>0.05) and 1-month all-cause mortality[0 (0/16) vs 4.2% (1/24), P>0.05]. The 6MWT, NT-proBNP, and NYHA were significantly improved at 1-month post TAVR compared with those before the procedure in the two groups (all P<0.05). Echocardiography showed significant reverse cardiac remodeling and improved left ventricular function compared with those before the procedure in the two groups. Conclusions:Transfemoral TAVR is a feasible and safe method for patients with pure native AR, and its short-term prognosis is similar to that in AS patients with well-established TAVR.

Objective:To analyze and screen the key genes of sepsis secondary to pulmonary infection by bioinformatics, and to provide theoretical basis for the basic research of the disease and find an ideal animal model program.Methods:Experiment 1 (bioinformatics analysis): gene expression data sets of pulmonary infection secondary sepsis patients and multiple sepsis animal models were screened by Gene Expression Omnibus (GEO) Database, and gene differences were analyzed by R software. Differential genes were analyzed by gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Correlation analysis was conducted between differential genes and clinical symptoms in the data set of pulmonary infection secondary sepsis, and the correlation heat map between differential genes and clinical symptoms was drawn. Key genes were screened by weighted gene co-expression network analysis (WGCNA) and protein-protein interaction network analysis (PPIN) clustering. Experiment 2 (sepsis animal model preparation): male mice weighing 21-25 g were randomly divided into the key genes group and the control (Sham) group. And cecal ligation and puncture (CLP) was used to establish mouse sepsis model, while the mice in sham group were performed by exposure of cecum. And all the mice were scarified 24 hours after surgery to extract the total RNA from lung tissue, real time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA expression of key genes.Results:Experiment 1 (bioinformatics analysis): 319 differential genes were showed by GSE 134364 and GSE 65682 data set analysis of pulmonary infection secondary sepsis. And there was no genetic difference between community acquired pneumonia (CAP) and hospital acquired pneumonia (HAP) in patients with pulmonary infection secondary to sepsis. Obvious differences existed between differential genes in animal models, and there was no common differential gene. Differential genes in patients and animal models were similarly enriched in GO function, mainly in cell differentiation, regulation of cell process, and regulation of cellular response to stimuli, there were significant differences in pathway enrichment, among which, CLP animal models showed higher consistency with patients. The key genes obtained by WGCNA and PPIN analysis were MAPK14, NLRC4 and LCN2. Experiment 2 (sepsis animal model preparation): animal experiment results showed that the mRNA expressions of MAPK14, NLRC4 and LCN2 in lung tissue of CLP model mice were significantly up-regulated compared with the sham group.Conclusions:MAPK14, NLRC4 and LCN2 are key genes involved in the regulation of biological processes of pulmonary sepsis secondary to infection, and are potential research directions of this disease. What's more, CLP animal model can better reflect the biological characteristics of patients with pulmonary infection secondary sepsis, and is one of the ideal animal model schemes for pulmonary infection secondary sepsis.

Objective To investigate the correlation between the vitreomacular adhesion (VMA) and exudative age-related macular degeneration (AMD).Methods A literature research was performed in PubMed,EMbase,Cochrane Library,CNKI and Wanfang database from January 2000 to December 2016.Case-control studies on the relationship between VMA or posterior vitreous detachment and exudative AMD were included in this analysis.Literature screening and data extraction were performed according to inclusion and exclusion criteria.The qualities of the literatures were evaluated according to the Newcastle-Ottawa Scale (NOS).Seven literatures were selected into meta-analysis.The NOS score was 9 points in 1 article,8 scores in 4 articles,7 points in 2 articles.A total of 947 eyes with exudative AMD,638 eyes with dry AMD,and 618 eyes with controls were included.The correlation between exudative AMD and VMA were analyzed using the software Review manager 5.3.Results The prevalence of VMA in exudative AMD eyes was higher than that in controls [odds ratio (OR)=2.14,95％ confidence interval (CI)=1.19-3.84,P=0.010] and dry AMD eyes (OR=2.24,95％CI=1.24-4.03,P=0.007).There was no difference in PVD prevalence among exudative AMD eyes,dry AMD eyes (OR=0.44,95％CI=0.16-1.20,P=0.110) and controls (OR=0.70,95％CI=0.41-1.18,P=0.180).Conclusion There is correlation between VMA and exudative AMD.

Objective To explore the MRI diagnosis characteristics of reversible splenial lesion syndrome (RESLES).Methods The clinical and imaging data of 12 cases of RESLES was analyzed retrospectively.MRI plain scan and DWI of brain were performed in all the cases.Apparent diffusion coefficient (ADC) value was obtained through post-processing function.Results 8 of 12 cases were children and 4 were adults.The splenium of corpus callosum of 12 cases showed round lesions,with slightly long T1 and slightly long T2 signal on MRI,high signal on DWI,and low signal on ADC map.The mean value of ADC of 12 cases was about 0.43× 10-3 mm2/s.The MRI reexamination was made after treatment in 12 cases,which showed the lesion of splenium of corpus callosum disappeared,and clinical syndromes were improved.Conclusion The MRI manifestation of RESLES is characterized by solitary lesion.The ADC value could accurately reflect the molecular diffusion information of the lesion.

To investigate the effect of the Ginkgo Biloba Extract (GBE) on the asthma and examine its possible mechanisms, 75 asthma patients were divided into 4 groups and the patients were respectively treated with fluticasone propionate for 2 weeks or 4 weeks, or treated with fluticasone propionate plus GBE for 2 weeks or 4 weeks. Fifteen healthy volunteers served as healthy controls. Sputum inhalation with inhaling hypertonic saline (4%-5%) was performed. Lung ventilatory function and forced expiratory volume in one second (FEV1) were measured. The numbers of different cells in induced sputum were calculated. The expression of PKCα in the cells was immunocytochemically detected and the percentages of positive cells in different cells were counted. Interleukin-5 (IL-5) in sputum supernatants was detected with enzyme-linked immunosorbent assay. The percentage of eosinophils, lymphocytes, PKCα positive inflammatory cells and the concentration of IL-5 in asthmatic patients were higher than those in the controls (P＜0.05), and the eosinophils, lymphocytes,positive expression of PKCα and the level of IL-5 were significantly decreased in asthmatic patients after they were treated with fluticasone propionate or fluticasone propionate plus GBE. However,they were still significantly higher than those of the controls. Compared to the group treated with glucocorticosteroid for 2 weeks, no significant decrease was found in the percentage of eosinophils,lymphocytes, PKCα positive inflammatory cells and the IL-5 in the supernatant of induced sputum.Compared with the group treated with glucocorticosteroid for 2 or 4 weeks, significant decrease in the same parameters was observed in the group treated with fluticasone propionate and GBE for 4 weeks. The IL-5 level in the supernatant of induced sputum was positively correlated with the percentage of PKCα-positive inflammatory cells and the percentage of eosinophils in the induced sputum in asthma patient groups respectively (n=150, r= 0.83, P＜0.01; n=150, r=0.76, P＜0.01). The FEV1 was negatively correlated with the percentage of PKCα-positive inflammatory cells and the IL-5 levels in supernatant of induced sputum in asthma patients respectively (n=150, r=-0.77,P＜0.01; n=150, r= -0.64, P＜0.01). It is concluded that GBE could significantly decrease the infiltration of inflammatory cells such as eosinophils and lymphocytes in the asthmatic airway and relieve the airway inflammation. GBE may decrease the activation of the PKCα in the inflammatory cells and thereby decrease the IL-5 level in induced sputum. GBE may be used as a complement to the glucocorticosteroid therapy for asthma.

This study sought to assess the biocompatibility of P(DA-SA)-Adriamycin, a new controlled-release chemotherapy system, in rabbit brain, and to examine its controlled release effect both in vitro and in vivo and its curative effects in vitro. The reaction of animal brain to the implanted P(DA-SA) or P(DA-SA)-Adriamycin was observed. The controlled-release profiles in phosphate buffer solutions and in rabbit brain were measured by UV spectrometry. Then, through flow cytometer, the rate of apoptosis in cultured glioma cells was tested. The reaction of rabbit brain to P(DA-SA) polymer was moderate and not significantly different from that to Gelfoam. The controlled-release rate of P(DA-SA)-Adriamycin in vitro and in vivo was stable and the duration of controlled-release of P(DA-SA)-Adriamycin spanned three weeks. The rate for apoptosis of glioma cells of P(DA-SA)-Adriamycin group was 69.9%, which was significantly higher than that of the control group. In conclusion, P (DA-SA)-Adriamycin controlled release chemotherapy system that bears curative effect has favorable controlled-release effect and good biocompatibility in rabbit brain. This system has potential value in treatment of malignant brain tumor.
Animals ; Antibiotics, Antineoplastic ; administration & dosage ; pharmacokinetics ; Apoptosis ; drug effects ; Brain ; metabolism ; Decanoic Acids ; metabolism ; Doxorubicin ; administration & dosage ; pharmacokinetics ; Drug Carriers ; metabolism ; Drug Implants ; Materials Testing ; Polyesters ; metabolism ; Rabbits ; Tumor Cells, Cultured

Objective To explore the effects of Sodium Nitroprusside(SNP) on apoptosis of airway smooth muscle cells(ASMCs) of asthmatic rats in vitro.Methods Ten Wister rats were selected to make the models of asthma.The effect of SNP on the survival rate of asthmatic rat airway smooth muscle cells was detected by MTT method.The apoptosis of cells was detected by TUNEL method and flow cytometry.Results Comparing with asthma group,the survival rate of ASMCs was decreased significantly in SNP plus asthma group by MTT method(P