Objective To establish a high-performance liquid chromatography method to detect empagliflozin and related substances in empagliflozin bulk drug and tablets,and to provide technical support for quality control and unified monitoring of empagliflozin bulk drug and its tablets.Methods A liquid chromatography development system with the full factorial design of experiments and the Box-Behnken model was used to screen and optimize the chromatographic parameters.Related substances were detected in empagliflozin API and empagliflozin tablets from different companies with the optimized chromatographic parameters.Results The optimized chromatographic parameters were obtained:Shim-pack GIST C18-AQ column(250 mm×4.6 mm,5 μm)was used,column temperature was 15 ℃,gradient elution with water-acetonitrile as mobile phase was as below,flow rate was 1.2 mL·min-1,detection wavelength was set at 224 nm.The specificity of the method is good,with recoveries ranging from 94.8％to 101.7％,and RSD ranging from 0.5％to 3.1％.The known single impurity in APIs and tablets is less than 0.05％,any other unknown single impurity is less than 0.06％,and the total amount of impurities are less than 0.3％.The related substances of supervised sampling are under good control.Conclusion The method is reliable and robust for determining related substances of empagliflozin and its tablets.

Objective To investigate the mechanism of action of Guizhi Fuling pill in treating chro-nic prostatitis(CP)using network pharmacology and molecular docking techniques.Methods Compo-nents of Guizhi Fuling pill were collected from the Traditional Chinese Medicines Systems Pharmacolo-gy Platform(TCMSP),and target information was obtained from the SwissTarget database.Targets for chronic prostatitis were screened from the GeneCards,OMIM,CTD,and DisGeNET disease data-bases.A protein-protein interaction(PPI)network was established and analyzed.Gene ontology(GO)functional annotation and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway en-richment analysis were performed using the DAVID database.The Cytoscape software was employed to construct an association network linking the components of Guizhi Fuling Pill,their targets,and the targets of chronic prostatitis.Molecular docking was conducted using AutoDock Vina software to verify the binding stability between the components of Guizhi Fuling pill and their targets.Results After screening and deduplication in the TCMSP database,76 components of Guizhi Fuling Pill were iden-tified,and 655 component targets were retrieved from the SwissTarget database.There were 190 intersecting targets between GuizhiFuling Pill and chronic prostatitis.GO analysis indicated that Guizhi Fuling Pill may treat chronic prostatitis by participating in processes such asapoptosis,ATP binding,and signal transduction.KEGG analysis suggested that Guizhi Fuling Pill can regulate pathways such as phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)and mitogen-acti-vated protein kinase(MAPK)to intervene in chronic prostatitis.Molecular docking data demonstra-ted that the components of Guizhi Fuling pill exhibited stable conformations with their targets.Con-clusion The components of Guizhi Fuling Pill can stably bind to their targets and exert therapeutic effects on chronic prostatitis through multiple targets and pathways.

Objective:To investigate the attention level of oral medicine residents in standardized training (referred to as residency training) to oral aesthetic information and its influencing factors.Methods:This study was a cross-sectional study, which included 262 residents undergoing standardized training at Hospital of Stomatology, Wuhan University in January 2020. Among them, there were 73 males and 189 females, with an age range of 23-33 (26±2) years. A questionnaire survey method was used, and the questionnaire included the basic characteristics of the subjects (gender, grade, major, identity, and having training experience in prosthodontic base or not) as well as their attention to oral aesthetic information (facial proportions, facial midline, smile line, facial contour, E-line, nasolabial angle, tooth shape and contour, and tooth proportions). Differences in the attention to oral aesthetic information among residents with different basic characteristics were compared, and a multivariable logistic regression was used to identify the influencing factors.Results:Among the 262 oral medicine residents, 256 (97.7%) believed that it was necessary to pay attention to oral aesthetic information. The differences in attention to facial proportions between residents of different genders and between those with and without training experience in the prosthodontic base were statistically significant (both P<0.05). The results of the multivariate logistic regression showed that male residents were less likely to pay attention to facial proportions compared with female residents ( OR=0.45, 95% CI: 0.23-0.88). Compared with residents specializing in orthodontics, non-orthodontic residents were less likely to pay attention to the E-line ( OR=0.14-0.27, 95% CI: 0.04-0.68) and nasolabial angle ( OR=0.14-0.31, 95% CI: 0.04-0.81). Residents who were graduate students with integrated four certificates were more likely to pay attention to the E-line compared with industry-based personnel ( OR=1.88, 95% CI: 1.01-3.49). Residents without training experience in the prosthodontic base were less likely to pay attention to the facial proportion ( OR=0.44, 95% CI: 0.20-0.97) and tooth shape and contour ( OR=0.33, 95% CI: 0.11-0.98) compared with those with such training experience in prosthodontic base. Conclusions:Oral medicine residents have a high level of attention to oral aesthetic information. Gender, major, identity, and having training experience in the prosthodontic base or not are factors that influence their attention to oral aesthetic information.

Objective To establish a high-performance liquid chromatography method to detect empagliflozin and related substances in empagliflozin bulk drug and tablets,and to provide technical support for quality control and unified monitoring of empagliflozin bulk drug and its tablets.Methods A liquid chromatography development system with the full factorial design of experiments and the Box-Behnken model was used to screen and optimize the chromatographic parameters.Related substances were detected in empagliflozin API and empagliflozin tablets from different companies with the optimized chromatographic parameters.Results The optimized chromatographic parameters were obtained:Shim-pack GIST C18-AQ column(250 mm×4.6 mm,5 μm)was used,column temperature was 15 ℃,gradient elution with water-acetonitrile as mobile phase was as below,flow rate was 1.2 mL·min-1,detection wavelength was set at 224 nm.The specificity of the method is good,with recoveries ranging from 94.8％to 101.7％,and RSD ranging from 0.5％to 3.1％.The known single impurity in APIs and tablets is less than 0.05％,any other unknown single impurity is less than 0.06％,and the total amount of impurities are less than 0.3％.The related substances of supervised sampling are under good control.Conclusion The method is reliable and robust for determining related substances of empagliflozin and its tablets.

Objective:To investigate the attention level of oral medicine residents in standardized training (referred to as residency training) to oral aesthetic information and its influencing factors.Methods:This study was a cross-sectional study, which included 262 residents undergoing standardized training at Hospital of Stomatology, Wuhan University in January 2020. Among them, there were 73 males and 189 females, with an age range of 23-33 (26±2) years. A questionnaire survey method was used, and the questionnaire included the basic characteristics of the subjects (gender, grade, major, identity, and having training experience in prosthodontic base or not) as well as their attention to oral aesthetic information (facial proportions, facial midline, smile line, facial contour, E-line, nasolabial angle, tooth shape and contour, and tooth proportions). Differences in the attention to oral aesthetic information among residents with different basic characteristics were compared, and a multivariable logistic regression was used to identify the influencing factors.Results:Among the 262 oral medicine residents, 256 (97.7%) believed that it was necessary to pay attention to oral aesthetic information. The differences in attention to facial proportions between residents of different genders and between those with and without training experience in the prosthodontic base were statistically significant (both P<0.05). The results of the multivariate logistic regression showed that male residents were less likely to pay attention to facial proportions compared with female residents ( OR=0.45, 95% CI: 0.23-0.88). Compared with residents specializing in orthodontics, non-orthodontic residents were less likely to pay attention to the E-line ( OR=0.14-0.27, 95% CI: 0.04-0.68) and nasolabial angle ( OR=0.14-0.31, 95% CI: 0.04-0.81). Residents who were graduate students with integrated four certificates were more likely to pay attention to the E-line compared with industry-based personnel ( OR=1.88, 95% CI: 1.01-3.49). Residents without training experience in the prosthodontic base were less likely to pay attention to the facial proportion ( OR=0.44, 95% CI: 0.20-0.97) and tooth shape and contour ( OR=0.33, 95% CI: 0.11-0.98) compared with those with such training experience in prosthodontic base. Conclusions:Oral medicine residents have a high level of attention to oral aesthetic information. Gender, major, identity, and having training experience in the prosthodontic base or not are factors that influence their attention to oral aesthetic information.

Purpose To investigate the expression of Che-mokine(C-X-C Motif)receptor 5(CXCR5)and its clinico-pathological significance in classic Hodgkin lymphoma(CHL).Methods The expression of CXCR5 was assessed in 33 pa-tients by immunohistochemistry(IHC),and retrospectively ana-lyzed the expression and clinical significance of CXCR5 in the four subtypes of CHL.Meanwhile,10 cases of ALK-positive an-aplastic large cell lymphoma(ALCL)and 10 cases of ALK-neg-ative ALCL were collected as the control group.ResultsThere were 31 cases with CXCR5-positive in all 33 cases(93.94％),including 15/16(93.75％)in nodular sclerosis CHL,12/13(92.31％)in mixed cellularity CHL,2/2 in lymphocyte-rich CHL,and 2/2 in lymphocyte-depleted CHL.The positive ex-pressions of CXCR5 in different immunophenotypes of CHL were as follow,31/33(93.94％)in CD30 positive and PAX5 weakly positive CHL.12/14(85.71％)in CD15 negative CHL,24/26(92.31％)in CD20 negative CHL,10/11(90.91％)in EBER-negative CHL and 5/6 in LMP1-negative CHL.CXCR5 were not expressed in all 20 cases of ALCL.Conclusion The positive expression rate of CXCR5 in CHL is high.When the tumor cells are negative for CD15,LMP1 and CD20 or EBER,CXCR5 also has a high positive expression rate,which is helpful for the diagnosis of CHL.CXCR5 can be used to differentiate CHL from ALCL,especially the cases lacking typical morpholo-gy and immunohistochemistry.

Objective:To evaluate the effect of activating adenosine A2B receptors on autophagy during myocardial ischemia-reperfusion (I/R) and the role of phosphatidylinositol 3-kinase/serine/threonine protein kinase (PI3K/Akt) signaling pathway in rats.Methods:Forty-eight clean-grade healthy male Sprague-Dawley rats, weighing 220-280 g, were divided into 4 groups ( n=12 each) using a random number table method: sham operation group (group Sham), myocardial I/R group (group I/R), adenosine A2B receptor agonist BAY 60-6583 group (group BAY) and BAY 60-6583+ PI3K inhibitor LY 294002 group (group BAY+ LY). Myocardial I/R was induced by occlusion of the anterior descending branch of the left coronary artery for 30 min followed by 120-min reperfusion.BAY 60-6583 1 mg/kg was intraperitoneally injected at 5 min before reperfusion in group BAY.BAY 60-6583 1 mg/kg was intraperitoneally injected at 5 min before reperfusion and LY 294002 10 mg/kg was intraperitoneally injected at 10 min before reperfusion in group BAY+ LY.Blood samples were obtained at the end of reperfusion for determination of concentrations of lactate dehydrogenase (LDH) and creatine kinase-MB (CK-MB) in serum (by enzyme-linked immunosorbent assay). The animals were sacrificed, and myocardial tissues were obtained for measurement of the percentage of myocardial infarct size (by Evan Blue and TTC double-staining) and for determination of the expression of microtubule-associated protein 1 light chain 3 (LC3Ⅰ), LC3Ⅱ, Beclin-1 and phosphorylated Akt (p-Akt) (by Western blot). The ratio of LC3Ⅱ/LC3Ⅰ was calculated. Results:Compared with group Sham, the serum LDH and CK-MB concentrations and percentage of myocardial infarct size were significantly increased, the expression of p-Akt was down-regulated, the expression of Beclin-1 and LC3Ⅱ was up-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was increased in group I/R ( P<0.05). Compared with group I/R, the concentrations of serum LDH, CK-MB and percentage of myocardial infarct size were significantly decreased, the expression of p-Akt was up-regulated, the expression of Beclin-1 and LC3Ⅱ was down-regulated, and the ratio of LC3Ⅱ/LC3Ⅰ was decreased in the group BAY ( P<0.05), and no significant change was found in the parameters mentioned above in group BAY+ LY ( P>0.05). Compared with group BAY, the concentrations of serum LDH, CK-MB and percentage of myocardial infarct size were significantly increased, the expression of p-Akt was down-regulated, the expression of Beclin-1 and LC3Ⅱ was up-regulated and the ratio of LC3Ⅱ/LC3Ⅰ was increased in group BAY+ LY ( P<0.05). Conclusion:Activating adenosine A2B receptors can decrease autophagy of myocardial cells during myocardial I/R injury, and the mechanism may be related to activating PI3K/Akt signaling pathway in rats.

Objective:To evaluate the role of endoplasmic reticulum stress in myocardial ischemia-reperfusion (I/R) injury and the relationship with autophagy in rats.Methods:Thirty-six healthy adult male Sprague-Dawley rats, weighing 250-300 g, were divided into 3 groups ( n=12 each) using a random number table method: sham operation group (Sham group), myocardial I/R group (IR group), and endoplasmic reticulum stress inhibitor 4-PBA group (PBA group). Myocardial I/R was produced by occlusion of left anterior descending branch of coronary artery for 30 min followed by reperfusion for 120 min.Sham group only underwent thoracotomy without block of left anterior descending branch of coronary artery.Endoplasmic reticulum stress inhibitor 4-PBA 500 mg·kg -1·d -1 was given intragastrically for 3 consecutive days before the I/R model was developed in PBA group, while the equal volume of normal saline was given instead in Sham and IR groups.The blood samples from the iliac vein were collected at 120 min of reperfusion for determination of the plasma creatine kinase isoenzymes (CK-MB) and cardiac troponin I (cTnI) concentrations (by enzyme-linked immunosorbent assay). The rats were then sacrificed, and myocardial tissues were removed for detection of myocardial glucose-regulated protein 78 (GRP78) and microtubule-associated protein 1 light chain 3 Ⅱ (LC3 Ⅱ) and autophagy-related protein 5 (ATG5) expression (by Western blot). Result:Compared with Sham group, the concentrations of CK-MB and cTnI in plasma were significantly increased in IR and PBA groups, the expression of GRP78, ATG5 and LC3Ⅱ was up-regulated, and the pathological damage was aggravated in IR group ( P<0.05). Compared with IR group, the concentrations of CK-MB and cTnI in plasma were significantly decreased, the expression of GRP78, ATG5 and LC3Ⅱ was down-regulated ( P<0.05), and the pathological changes were significantly attenuated in PBA group. Conclusion:Endoplasmic reticulum stress is involved in the process of myocardial I/R injury, and the mechanism may be related to promotion of autophagy in rats.

Objective:To explore the association of peripheral blood neutrophil lymphocyte ratio(NLR) with primary osteoporosis (POP) in Chinese elderly women.Methods:From December 2015 to April 2019, a total of 788 elderly women in Chongqing Three Gorges Central Hospital were included in this study.They were divided into three groups according to bone mineral density: 242 cases of osteopenia, 206 cases of normal bone mineral density and 340 cases of osteoporosis.Their height, weight, statuses of bone of left femoral neck and results of blood tests were recorded, and the values of BMI, NLR and OSTA were calculated.The SPSS statistics software was used to analyze the differences of parameters among the three groups.Logistic regression analysis was used to get significant independent related factors for osteoporosis.The relationship of NLR and OSTA with T-score of left femoral neck was confirmed by Pearson correlation test.ROC curves were used to define the values of NLR and OSTA in diagnosis of osteoporosis.Results:There were statistically significant differences in age(median values: 66, 68 and 70, χ 2=71.596, P<0.001), BMI(median values: 23.46, 24.04 and 25.415, χ 2=64.936, P<0.001) and NLR(median values: 1.97, 2.435 and 2.84, χ 2=106.218, P<0.001) among the three groups(all P<0.01). Logistic regression analysis demonstrated that age, BMI and NLR were all independent significant related factors.There was correlation of NLR, OSTA with T-score of left femoral neck.In diagnosis of osteoporosis, the cut-off value, maximum Youden index, sensitivity and specificity were 2.395, 0.299, 70.3%, 59.6% for NLR, and -1.315, 0.174, 73.6%, 43.5% for OSTA. Conclusion:NLR has intermediate sensitivity and specificity in screening POP in Chinese elderly women, and deserves future researches.

Objective:To construct and identify the lentiviral vector of adenosine RNAi-adenosine A2a receptor (A2aR) in rats.Methods:Three pairs of short hairpin RNA(shRNA)-A2aR sequences (shRNA-A2aR 1, shRNA-A2aR 2, shRNA-A2aR 3) were designed, and three pairs of double-stranded shRNA oligos were respectively inserted into the shRNA virus vector to gain three kinds of shRNA lentiviral recombinant plasmid.The recombinant plasmid, packaging vector, and shuttle vector were co-transfected into 293T cells to obtain virus liquid.The experiment was performed in two parts.Part Ⅰ The rat primary cardiomyocytes were divided into 3 groups ( n= 6 each) by a random number table method: vehicle group (V group), shRNA-A2aR 1 group and shRNA-A2aR 3 group.Each group was transfected with virus solution of MOI 10 for 48 h. The expression of A2aR was detected by Western blot to select the most efficient lentivirus vector.Part Ⅱ The cardiomyocytes were randomly divided into 6 groups ( n=36 each): vehicle group (V group), MOI5 group, MOI10 group, MOI15 group and MOI20 group.Each group was transfected with the corresponding MOI virus liquid (the most effective lentivirus vector). At 24, 48, and 72 h of transfection, the cell viability and cell death were observed with a fluorescent microscope, and the A2aR expression was detected by Western blot to determine the interference efficiency. Results:Part Ⅰ Two types of shRNA-A2aR lentiviral vectors (shRNA-A2aR 1, 3) were successfully constructed, among which shRNA-A2aR 3 virus solution with a titer of 3.5×10 8 TU/ml had the best effect.Compared with group V and group shRNA-A2aR 1, the expression of A2aR in cardiomyocytes was significantly down-regulated ( P<0.01), and the interference efficiency of shRNA-A2aR 3 was 73% in shRNA-A2aR 3 group.Part Ⅱ shRNA-A2aR 3 was selected to screen out the transfection plan.The cell survival rate in each group was more than 85% at 24 h of transfection, the cell survival rate was more than 80% at 48 h of transfection in MOI5 and MOI10 groups; the cell survival rate in each group was less than 70% at 72 h of transfection.Under an inverted fluorescent microscope, a slightly lower fluorescence density was found in MOI5 group, the fluorescent density was higher and the cell condition was better at 48 h of transfection in MOI10 group and at 24 h of transfection in MOI20 group, and the cardiomyocyte viability was significantly decreased, and dead cells were increased at 72 h of transfection in each group.The results of Western blot showed that the interference efficiency at 48 h of transfection in MOI10 group, 48 h in MOI15 group, 24 and 48 h in MOI20 group was all > 70%. Conclusion:MOI of 10, transfection for 48 h or MOI of 20, transfection for 24 h is the optimal transfection protocol.