Objective To determine the effectiveness and safety of prophylactic intravenous ibuprofen compared to placebo/no intervention on the prevention of patent ductus arteriosus(PDA) in preterm infants.Methods Randomized controlled trials(RCT) or quasi-RCT comparing prophylactic intravenous ibuprofen versus placebo/no treatment for prevention of PDA in preterm infants were enrolled. The standard search strategy included electronic search and manual search. Electronic search was carried out in databases including PubMed,ScienceDirect, EMBASE, OVID, Cochrane Library, VIP Chinese Periodical Database and Chinese Digital Hospital Library (www. chkd. cnki. net) without language restriction. As a supplementation,references in previous reviews and studies identified as relevant had been examined by manual search. RevMan 5.0. 21 was used in the statistical analysis. Effects were expressed as weighted mean difference (WMD) and 95% confidence interval (CI) for continuous data while risk ratio (RR) and 95%CI for categorical data.Results Four studies qualified for this meta-analysis including three graded 4 and one graded 3 with Jadad scale. Prophylactic use of ibuprofen significantly decreased the incidence of PDA on day three (RR=0. 40,95 % CI: 0. 31-0. 51, P＜0.01 ), the need for rescue treatment with cyclo-oxygenase inhibitors (RR = 0. 18,95 % CI:0. 07-0. 45, P = 0. 0003) and the need for surgical ligation (RR = 0. 34,95 % CI: 0. 14-0. 81, P =0. 02) in the prophylactic group. No significant difference of pulmonary, cerebral, gastrointestinal, and renal complications were found between the prophylactic and control groups. Conclusions Prophylactic intravenous ibuprofen significantly reduces the risk of PDA on day three, decreases the need for rescue treatment with cyclo-oxygenase inhibitors and surgical ligation without significant pulmonary, cerebral,gastrointestinal, and renal complications were seen in the prophylactic group. Due to the limits of evidences to date, prophylactic ibuprofen intravenously for prevention of PDA in preterm infants is not recommended.

ObjectiveTo investigate the therapeutic effect and function mechanism of ginkgo biloba extract (EGb) for severe acute pancreatitis.MethodsFifty - four patients,who were diagnozed according to the inclusion criteria,were divided into two groups at random.They were from the People's Hospital of Anyang City from November 2006 to December 2010.In treatment group,25 patients were treated with integrated EGb and other comprehensive treatment group,and 21 patients completed the treatment; in the the contrast group,the 29 patients were trested with simple comprehensive treatment group,and 23 patients completed the treatment. Comprehensive therapy included Somatostatin and antibiotic,ect. Besides the comprehensive therapy,patients in the treatment group were intra - venously infused with 87.5 mg EGbeveryday.Before the day of treatment,on the 3rd and 7th day after the treatment,blood TNF-α,IL-6 and amylase level were determined.Blood MDA level was measured and Balthazar CT grades was observed before the day of treatment and on the 7th day after the treatment.Abdominal pain,abdominal distension and tenderness were observed.Pancreatic infection incidence rate was compared between the two groups.All experimental data were processed with SPSS version 11. 0 statistical software. Quantitative data were analyzed byRepeated Measurement ANOVA or t - test.Count data were analyzed by x2 test.ResultsThe values of blood TNF-α,IL-6 and amylase level were significantly lower in treatment group than in contrast group on the 3rd and 7th day after the treatment ( P ＜ 0.05 ).The values of blood MDA and Balthazar CT grades were significantly lower in treatment group than in the contrast group on the 7th day after the treatment (P ＜ 0.05 ).There was a significantly shorter period of abdominal pain,abdominal distension and tenderness in treatment group than in contrast group.( P ＜ 0.01 ).The pancreatic infection incidence rate of the treatment group was lower than that of the contrast group ( P ＜ 0.05 ).ConclusionsEGb could alleviate notably the cytokine release,restrain oxidative stress and remove the free oxygen factor,prevent and treat Pancreatic infection and necrosis.

Objective To investigate the effects of advanced glycation end products(AGE)on the expressions and activity of cathepsin D(CatD)in ultraviolet A(UVA)?irradiated human dermal fibroblasts. Methods Human dermal fibroblasts were isolated and harvested from the circumcised foreskin of children, and subjected to a primary culture. CCK?8 assay was performed to screen non?cytotoxic concentrations of AGE?bovine serum albumin (BSA). Some fibroblasts were incubated with 50, 100 and 300 mg/L AGE?BSA separately for 24 hours, with untreated cells as the control group. Then, reverse transcription(RT)?PCR, Western?blot analysis and a fluorimetric assay were performed to measure the mRNA and protein expressions as well as activity of CatD, respectively. Some fibroblasts were classified into six groups: control group receiving no treatment, AGE?BSA group and BSA group treated with the highest non?cytotoxic concentration of AGE?BSA and the same concentration of BSA respectively for 24 hours, UVA group irradiated by 10 J/cm2 UVA, UVA?AGE?BSA group and UVA?BSA group treated with AGE?BSA and BSA at the above non?cytotoxic concentration respectively for 24 hours both before and after UVA radiation at 10 J/cm2. After the treatments, RT?PCR, Western?blot analysis and a fluorimetric assay were conducted to detect mRNA and protein expressions and activity of CatD respectively. Results AGE?BSA of 50- 200 mg/L exhibited no obvious influence on cellular proliferation of fibroblasts. The fibroblasts incubated with AGE?BSA of 50, 100 and 200 mg/L showed a significant increase in the mRNA expression(0.267 ± 0.007, 0.348 ± 0.007, and 0.418 ± 0.006 respectively), protein expression (1.403 ± 0.181, 2.233 ± 0.090 and 2.477 ± 0.111 respectively), and activity(1.760 ± 0.080, 2.330 ± 0.060 and 2.890 ± 0.080 respectively)of CatD compared with the control group(mRNA:0.161 ± 0.006;protein:0.903 ± 0.200;activity:1.100 ± 0.090, all P < 0.05). AGE?BSA increased CatD expressions and activity in a dose?dependent manner. The mRNA and protein expressions as well as activity of CatD were significantly higher in the UVA group than in the control group (mRNA expression: 0.480 ± 0.005 vs. 0.155 ± 0.005; protein expression: 2.583 ± 0.199 vs. 0.920 ± 0.235;activity:2.970 ± 0.110 vs. 1.110 ± 0.040, all P<0.05), but significantly lower in the UVA?AGE?BSA group than in the UVA group(mRNA expression:0.394 ± 0.008 vs. 0.480 ± 0.005;protein expression:2.070 ± 0.125 vs. 2.583 ± 0.199;activity: 2.560 ± 0.060 vs. 2.970 ± 0.110, all P < 0.05). Conclusion AGEs could increase CatD expressions and activity in human dermal fibroblasts not receiving UVA irradiation, but inhibit their increase in UVA?induced human dermal fibroblasts.

Objective:To explore the structural characteristics of intestinal microflora in children with non-alcoholic fatty liver disease（NAFLD）and the relationship between intestinal microflora and the occurrence as well as development of NAFLD in children.Methods:Fifteen children with NAFLD diagnosed at the First People's Hospital of Yunnan Province from January 2022 to December 2022 were selected as subjects，and 15 healthy children who received routine physical examinations at the outpatient clinic during the same period were randomly selected as healthy control group.The height，weight，waist circumference，blood pressure，blood biochemistry of all children were collected.At the same time，the fresh fecal samples of all children were collected，and the biological information of intestinal flora was analyzed by 16S rRNA sequencing.Results:In the NAFLD group，there were eight males and seven females，with an average age of（11.13±1.77）years.In healthy control group，there were seven males and eight females，with an average age of（9.73±2.25）years.There were no significant differences in sex，age，blood pressure between two groups.Compared with the healthy control group，the levels of body mass index，waist circumference，waist-to-height ratio，alanine aminotransferase，aspartate transaminase，gamma-glutamyl transpeptidase，total bilirubin，direct bilirubin，unconjugated bilirubin，low density lipoprotein cholesterol，uric acid and serum insulin significantly increased and high density lipoprotein cholesterol significantly decreased in NAFLD group（ P<0.05）.The results of species diversity analysis showed that chaol index and observed-species index in NAFLD group were significantly higher than those in healthy control group（ P<0.05）.Species diversity analysis showed that the species with increased abundance in NAFLD group included：Proteobacteria，Gammaproteobacteria，Enterobacteriaceae，Klebsiella，Escherichia-Shigella，Escherichia-Shigella-unclassified.Differential species with increased abundance in the healthy control group included：Bifidobacterium species，Bifidobacterium，Bifidobacteriaceae，Bifidobacteriales，Actinobacteria，Actinobacteriota，Bacteroidia，Bacteroidales，Streptococcus-thermophilus. Conclusion:There are metabolic abnormalities and obvious changes in the structure of intestinal flora in children with NAFLD.Exogenous supplementation of Bifidobacterium，Streptococcus thermophilus and Bacteroides may prevent the occurrence of NAFLD，delay the progression of disease and improve fat deposition in the liver.

Objective To evaluate the effect of photoaging on the degradation of advanced glycation end products (AGEs) by human dermal fibroblasts.Methods Some cultured human dermal fibroblasts were subjected to repetitive ultraviolet A (UVA) radiation (UVA radiation group) to establish a photoaging cell model,which was then evaluated by cell counting kit 8 (CCK-8) assay,senescenceassociated β-galactosidase staining and detection of apoptosis rate.Moreover,fibroblasts receiving no treatment served as control group.Some other primary fibroblasts were divided into 4 groups:photoaged group receiving UVA radiation,non-photoaged group receiving no treatment,AGE-treated photoaged group treated with UVA radiation followed by the treatment with 200 mg/L AGE-bovine serum albumin (BSA),and AGE-treated non-photoaged group treated with 200 mg/L AGE-BSA alone.After the treatment with AGE-BSA for 4-72 hours,flow cytometry was performed to determine the fluorescence intensity of AGE-BSA in fibroblasts of the above groups.After 8-hour treatment with AGE-BSA,confocal laser scanning microscopy was performed to localize and semiquantitatively detect AGE-BSA in fibroblasts,and enzymelinked immunosorbent assay (ELISA) was conducted to detect AGE-BSA levels in fibroblasts,as well as changes in the intracellular AGE-BSA level within 24 hours after the removal of AGE-BSA.Results Compared with the control group,the UVA radiation group showed significantly decreased cellular proliferative activity (t =7.559,P ＜ 0.05),but significantly increased apoptosis rate and percentage of β-galactosidase-positive fibroblasts (t =14.075,43.524 respectively,both P ＜ 0.05).Flow cytometry revealed that the average fluorescence intensities of AGE-BSA after 4-,8-,16-,24-,48-and 72-hour treatment with AGE-BSA were significantly higher in the AGE-treated photoaged group (293.00 ± 8.19,359.67 ± 11.59,347.00 ± 12.29,338.00 ± 12.77,334.67 ± 14.22 and 336.30 ± 10.21,respectively) than in the photoaged group (all P ＜ 0.05),as well as in the AGE-treated non-photoaged group (222.33 ± 8.74,276.33 ± 6.11,256.33 ± 5.51,243.00 ± 10.15,236.33 ± 1.53 and 240.33 ± 1.52,respectively) than in the non-photoaged group (all P ＜ 0.05).Moreover,the average fluorescence intensities of AGE-BSA at different time points were all significantly higher in the AGE-treated photoaged group than in the AGE-treated non-photoaged group (all P ＜ 0.05).Confocal laser scanning microscopy showed that AGE-BSA was mainly localized in lysosomes after endocytic uptake into the fibroblasts,and the AGE-treated photoaged group showed significantly increased fluorescence intensity of AGE-BSA compared with the AGE-treated non-photoaged group (P ＜ 0.05).ELISA revealed that the intracellular AGE level in the AGE-treated non-photoaged group at 24 hours after the removal of AGE-BSA was decreased by (14.6 ± 1.2)％ compared with that before the removal,and the degradation rate of AGE-BSA was significantly higher in the AGE-treated non-photoaged group than in the AGE-treated photoaged group (7.6％ ± 1.4％,t =6.604,P ＜ 0.05).Conclusion The internalized AGE-degradating ability decreases in photoaged fibroblasts,which may induce the accumulation of AGEs in photoaged skin.

Objective To determine the expression of cathepsin D and advanced glycation end products (AGEs)in skin tissues from patients of different ages or skin tissues with different degrees of sun exposure,to evaluate their correlation,and to preliminarily investigate the role of cathepsin D in the degradation and accumulation of AGEs in photoaged skin.Methods Skin tissues were collected from sunexposed and sun-protected body sites in patients aged 15-20 years,35-40 years,55-60 years or 75-80 years.These skin tissues were divided into 8 groups according to age of patients and degrees of sun exposure,and there were 6 specimens in each group.Immunohistochemical and immunofluorescent methods were used to measure the expression of cathepsin D and AGEs in the skin tissues.Statistical analysis was carried out by factorial design analysis of variance,Wilcoxon rank sum test and Kruskal-Wallis rank sum test for analyzing associations of the expression of cathepsin D and AGEs with age and sun exposure,as well as by Pearson correlation analysis for assessing the correlation between cathepsin D expression and AGEs expression.Results Immunohistochemical study showed that the expression of cathepsin D markedly decreased along with the increase of age,but the accumulation of AGEs gradually increased along with the increase of age.In the same age group,the cathepsin D expression was lower in the sun-exposed skin tissues than in the sun-protected skin tissues,while the accumulation of AGEs was more in the sun-exposed skin tissues than in the sun-protected skin tissues.Factorial design analysis of variance showed that sun exposure could decrease the expression of cathepsin D (F =58.70,P ＜ 0.001),but increase the accumulation of AGEs (F =158.18,P ＜ 0.001).Moreover,the increase of age could lead to decreased expression of cathepsin D (F =79.49,P ＜ 0.001),and increased expression of AGEs (F =106.06,P ＜0.001).Compared with the sun-protected skin tissues,the sun-exposed skin tissues in all the age groups showed significantly lower absorbance value of cathepsin D (35-40 years:0.020 ± 0.005 vs.0.032 ± 0.005;55-60 years:0.012 ± 0.004 vs.0.026 ± 0.002;75-80 years:0.002 ± 0.001 vs.0.013 ± 0.004;all P ＜0.001),but higher absorbance value of AGEs (35-40 years:0.030 ± 0.008 vs.0.010 ± 0.003;55-60years:0.066 ± 0.010 vs.0.021 ± 0.004;75-80 years:0.085 ± 0.015 vs.0.035 ± 0.009;all P ＜ 0.001)except the age group of 15-20 years.No matter whether the skin tissues were sun-exposed or sunprotected,there were significant differences in the expression of cathepsin D and AGEs among different age groups (all P ＜ 0.001).The results of double immunofluorescence staining were similar to those of immunohistochemical study.Pearson correlation analysis showed that the expression of cathepsin D in the sun-exposed skin tissues was highly negatively correlated with the accumulation of AGEs (r =-0.915,P ＜0.05),while they were moderately negatively correlated in the sun-protected skin tissues (r =-0.730,P ＜0.05).Conclusions Along with the increase of age,the expression of cathepsin D in skin tissues decreased,but the expression of AGEs increased.In the sun-protected skin tissues,the expression of cathepsin D was moderately negatively correlated with the expression of AGEs,while they were highly negatively correlated in the sun-exposed skin tissues,suggesting that cathepsin D may play an important role in the degradation and accumulation of AGEs in photoaged skin.

Objective To investigate the regulatory role of cathepsin D (CatD) in the degradation of intracellular advanced glycation end products (AGEs) endocytosed by human dermal fibroblasts (HDFs).Methods Cultured HDFs were treated with 1 μnol/L CA074Me (an inhibitor of CatB and CatL),75 μmol/L pepstatin A (an inhibitor of CatD) and 1 μmol/L MG-132 (an inhibitor of20S proteasome) separately for 4 hours,and then cell counting kit 8 (CCKS) assay and fluorometric assay were performed to determine the cellular viability and protease activity,respectively.The cells in the CA074Me group,pepstatin A group and MG-132 group were additionally treated with AGE-bovine serum albumin (BSA) for 8 hours,and the cells in the blank control group were treated with phosphate-buffered saline (PBS) alone.After 8-hour cultivation,the cells in the above groups were subsequently reincubated with fresh culture medium containing the corresponding inhibitors for 24 hours.Then,flow cytometry was performed to assess the mean fluorescence intensity of intracellular AGE-BSA at different time points.Some other HDFs were treated with 37.5,75 and 150 μmol/L pepstatin A and PBS separately for 4 hours,and then the cells in the 4 groups were treated with 200 mg/L AGE-BSA for 8 hours,followed by the removal of AGE-BSA from the medium and the treatment with 37.5,75 and 150 μmol/L pepstatin A and PBS respectively.Enzyme-linked immunosorbent assay (ELISA) was conducted to measure the mean concentration of intracellular AGE-BSA at different time points,and the degradation rate of AGE was calculated.Some HDFs were divided into 3 groups:blank control group receiving no treatment,NC group transfected with an empty vector,and CatD group transfected with a CatD-overexpressing lentiviral vector.Fluorescence microscopy was conducted to estimate the transfection efficiency.Reverse transcription-PCR,Western blot analysis and fluorometric assay were performed to determine the mRNA and protein expression,and activity of CatD respectively.Then,the cells in the above 3 groups were incubated with AGE-BSA for 8 hours,followed by the removal of AGE-BSA from the medium and the treatment with fresh culture medium.The detection methods were same as the above experiment,and the degradation rate was calculated.Results The cellular proliferative activity in the 1-μmol/L CA074Me group,75-μmnol/L pepstatin A group and 1-μ mol/L MG-132 group was more than 90％,and there was no significant difference between the 3 groups and the control group (100％,F =1.525,P ＞ 0.05).Twenty-four hours after the removal of AGE-BSA from the medium,the fluorescence intensities of intracellular AGE-BSA in the CA074Me + AGE-BSA group (275.00 ± 10.15) and MG-132 + AGE-BSA group (259.00 ± 11.14) significantly decreased compared with those at the 8-hour time point (295.00 ± 6.56 and 285.67±8.74 respectively;paired t test,t =4.778,6.154 respectively,both P ＜ 0.05),while no significant difference was observed in the fluorescence intensities of intracellular AGE-BSA in the pepstatin A + AGE-BSA group between the 8-hour time point and 32-hour time point (P ＞ 0.05).The degradation rates of intracellular AGE-BSA within 24 hours in the 37.5,75 and 150 μmol/L pepstatin A groups were 9.64％ ± 1.27％,5.62％ ± 0.47％ and 3.21％ ± 0.73％ respectively;there were significant differences among the 3 groups (F =45.876,P ＜ 0.05),and the degradation rate significantly decreased along with the increase of pepstatin A concentration (P ＜ 0.05).Fluorescence microscopy showed no fluorescent cells in the blank control group,while the NC group and CatD group both showed a high proportion (＞ 80％) of fluorescent cells.The mRNA and protein expression as well as the activity of CatD were significantly higher in the CatD group than in the blank control group and NC group (all P ＜ 0.05).The CatD + AGE-BSA group showed a significantly higher degradation rate of intracellular AGE-BSA within 24 hours compared with the AGE-BSA group and NC + AGE-BSA group (both P ＜ 0.05).Conclusion CatD can promote the degradation of intracellular AGE-BSA endocytosed by HDFs.