Objective:To preliminarily investigate the effect of circIGF2BP3 on autophagy in photoaged dermal fibroblasts.Methods:Human dermal fibroblasts were isolated from circumcised foreskin tissues from 6 children in the Department of Urological Surgery, Third Affiliated Hospital of Sun Yat-sen University. An ultraviolet A (UVA) -induced photoaged human dermal fibroblast model (UVA radiation group) was established by repeated UVA radiation at a dose of 10 J/cm 2 for 14 consecutive days, and human dermal fibroblasts receiving no treatment served as control group. The photoaged cell model was verified by β-galactosidase staining, Western blot analysis for determining P21 protein expression, and cell counting kit-8 (CCK8) assay for evaluating cell viability. Moreover, Western blot analysis was performed to determine the protein expression of autophagy-related proteins P62, microtubule-associated protein 1 light chain 3 (LC3) -Ⅰand LC3-Ⅱ in photoaged human dermal fibroblasts, and real-time quantitative RCR (qRT-PCR) to verify the differential expression of circIGF2BP3 between photoaged and normal human dermal fibroblasts. Furthermore, circIGF2BP3 was biologically annotated. Some cultured primary human dermal fibroblasts were divided into 4 groups: empty vector group transfected with an empty vector, UVA + empty vector group transfected with an empty vector followed by repeated UVA radiation, circIGF2BP3 group transfected with a circIGF2BP3-overexpressing lentiviral vector, UVA + circIGF2BP3 group transfected with a circIGF2BP3-overexpressing lentiviral vector followed by repeated UVA radiation. Western blot analysis was performed to determine the expression of autophagy-related proteins. Statistical analysis was carried out by using t test, one-way analysis of variance and least significant difference- t test. Results:Compared with the control group, the UVA radiation group showed significantly increased proportions of β-galactosidase-positive cells (61.33% ± 5.78% vs. 6.37% ± 0.32%, t = 9.49, P < 0.01) and P21 expression (1.25 ± 0.03 vs. 1.00 ± 0.05, t = 4.26, P < 0.05), but significantly decreased cell viability (74.33% ± 3.48% vs. 100%, t = 7.38, P < 0.01). Moreover, the P62 expression and LC3-Ⅱ/Ⅰ ratio were significantly higher in the UVA radiation group than in the control group (both P < 0.05). The relative expression of circIGF2BP3 was 0.72 ± 0.04 in the photoaged human dermal fibroblasts, which was significantly lower than that in the normal human dermal fibroblasts (1.00 ± 0.03, t = 5.46, P < 0.01). The P62 expression and LC3-Ⅱ/Ⅰ ratio were significantly lower in the circIGF2BP3 group (0.60 ± 0.01, 0.71 ± 0.01, respectively) than in the empty vector group (1.00 ± 0.02, 1.00 ± 0.01, t = 16.25, 2.75, P < 0.01, < 0.05, respectively), and lower in the UVA + circIGF2BP3 group (1.05 ± 0.02, 2.04 ± 0.05, respectively) than in the UVA + empty vector group (1.31 ± 0.02, 2.72 ± 0.14, t = 10.493, 6.472, respectively, both P < 0.01) . Conclusion:circIGF2BP3 can regulate autophagy in UVA-induced photoaged dermal fibroblasts, which provides a new potential therapeutic target for the prevention and treatment of skin photoaging.

Objective: To understand the etiology of hepatopathy of unknown etiology in patients undergoing liver biopsy. Methods: Demographic data and pathological examination reports of patients with hepatopathy of unknown etiology who underwent liver biopsy examination at outpatient and inpatient of the Second Hospital of Nanjing between January 2017 and June 2018 were retrospectively collected. All liver histopathological sections combined with clinical and pathological features based on liver biopsy examinations were diagnosed by a reputed clinician and a pathologist. Results: A total of 470 cases with hepatopathy of unknown etiology who underwent liver biopsy were enrolled. Of these, 425 cases (90.4%) had a definite diagnosed disease after comprehensive analysis of pathological and clinical data. The diagnosis of hepatopathy of unknown etiology included 11 diseases: 90 cases with autoimmune hepatitis had autoimmune liver disease (19.1%), 38 cases had primary biliary cholangitis (8.1%), 43 cases with overlap syndrome of autoimmune hepatitis had primary biliary cholangitis (9.1%), 118 cases had drug-induced liver injury (25.1%), 75 cases had nonalcoholic fatty liver disease (NAFLD) (16.0%), 12 cases had alcoholic liver disease (2.6 cases) %), 15 cases (3.2%) had vascular liver disease, 7 cases (1.5%) had hereditary metabolic liver disease, 5 cases (1.1%) had other systemic diseases, 16 cases (3.4%) had more than two kinds of liver diseases, and 6 cases (1.3%) had others rare liver diseases. Conclusion Over 90% cause of the hepatopathy of unknown etiology in the long run can be determined, and the main causes are autoimmune liver disease, drug-induced liver injury, and nonalcoholic fatty liver disease, which needs multidisciplinary cooperation to diagnose, and clinicians need to master the basic and clinical knowledge of liver diseases as well as liver pathology, hepatobiliary imaging, and genetics.

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.

Objective To establish fingerprint of volatile components in golden throat lozenges by GC-MS. Methods Volatile components from 10 batches of golden throat lozenges were extracted by steam distillation method and analyzed by GC-MS with n-tetradecane as internal standard. Fingerprint peak detection and similarity evaluation were applied to the total ion chromatogram(TIC)of GC-MS by "Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (Version 2004)" ,fingerprint peak was identified by mass spectrometry. Results A GC-MS fingerprint was established based on 10 common peaks as characteristic fingerprint information.The similarity of fingerprint peaks from the 10 batches of samples were more than 0.998.Ten fingerprint peaks were determined by mass spectrometry, all of which were composed of monoterpenes and monoterpenes containing oxygen, the highest content of which was L-menthol containing oxygen monoterpenes, accounting for 83.17% of the total. Conclusion The fingerprint established by gas chromatography-mass spectrometry can completely reflect the volatile components of golden throat lozenges tablets, with strong characteristics and specificity, and can be used as an effective method for the quality control of volatile components inf golden throat lozenges.

Objective To investigate the impacts of water supply on water quality in the pipeline of the dental comprehensive treatment platform to contribute to pollution control.Methods Totally 8 platforms from the stomatological department underwent 2-a detection and tracing.The water sources included tap water,distilled water and filtered water,and the discharge water went through sampling and bacteriological analysis before and after disinfection.Results The mean numbers of colonies by tap water,distilled water and filtered water were (472±385),(380±372) and (446±382) cfu/ml respectively,and the qualification rates by tap water,distilled water and filtered water were 33.3％,45.8％ and 37.5％ respectively.All the colonies numbers were limited within 0 and 60 cfu/ml with the qualification rate being 100％ after disinfection.Conclusion Water source cannot relieve bacterial infection effectively,and disinfection eliminates bacteria and improves water quality in the pipeline of the dental comprehensive treatment platform.

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 investigate the effects of four interior-warming drugs( galangal,cinnamon,evodia rutaecarpa,and dried ginger)on the tension of ileum smooth muscle and Ca2+-ATPase on the cell membrane in rabbits. Methods The effects of galangal,cinnamon,evodia rutaecarpa,and dried ginger were examined on normal ileum smooth muscle,in vitro intestinal muscle contraction caused by acetylcholine(ACh),barium chloride(BaCl2 )and histamine(His), and ACh-induced calcium release by using BL-420E+ biological signal collection and processing system.The average tension was measured within 1 min before delivery and within 3 minutes after the treatment,and the inhibition rate was calculated according to the average tension value.The effects of sera containing galangal,cinnamon,evodia rutaecarpa,and dried ginger on Ca2+-ATPase activity on the cell membrane of the intestinal smooth muscle were examined by phosphorus method. Results Galangal,cinnamon,evodia rutaecarpa,and dried ginger at high concentrations could restrain in vitro intestinal contraction in normal circumstances(P<0.05 or P<0.01).Significant inhibitory effects on intestinal contraction caused by ACh,His and BaCl2 were found in low,medium and high concentration groups(P<0.01).There was a dose-effectiveness relationship between the inhibition rate and final drug concentrations.The ACh-induced intracellular and extracellular calcium dependent contraction were significantly inhibited by the four interior-warming drugs( P < 0. 05 or P < 0. 01). The Ca2+-ATPase activities were( 0. 384 ± 0.070),(0.302±0.016),(0.307±0.016),(0.296±0.016),(0.313±0.003)U·mg-1 ,respectively,in intestinal smooth muscle in normal control group and high concentration groups of galangal,cinnamon,evodia rutaecarpa,and dried ginger(P<0.01). Conclusion Interior-warming drugs may relax intestinal smooth muscle by reducing the intracellular calcium release and the extracellular calcium inflow via receptor-controlled calcium channels,and inhibiting the Ca2+-ATPase activity in smooth muscle.

BACKGROUND:Borosilicate cannot only be mineralized to form hydroxy carbonate apatite layer, but also have strong chemical reactivity to promote bone cel regeneration. OBJECTIVE:To investigate the effect of the borosilicate bioglass on the growth behavior of rabbit osteoblasts through in vitro culture experiment. METHODS:The initial and secondary extracts of borosilicate bioglass were prepared according to the requirement of ISO10993-12: 2007. The bone marrow mesenchymal stem cels of rabbits were isolated and cultured. The second generation bone marrow mesenchymal stem cels were induced to differentiate into osteoblasts. The osteoblasts of the 5th-15th RESULTS AND CONCLUSION:The osteoblasts proliferation in the initial extract and secondary extract groups was better than that in the α-MEM medium group (P < 0.05). The osteoblasts proliferation in the initial extract group was better than that in the secondary extract group (P < 0.05). The total protein content of osteoblasts in the initial extract group was higher than that in the secondary extract and α-MEM medium group (P < 0.05). There were no significant differences in the alkaline phosphatase activity, apoptosis rate, horizontal migration distance of osteoblast and transmembrane cel number in Transwel between these three groups. These results demonstrate that borosilicate bioglass has good biocompatibility and has a certain benign regulatory role in generations were obtained and cultured with the initial and secondary extracts of borosilicate bioglass and α-MEM medium, respectively. The effects of borosilicate bioglass on the osteoblasts proliferation, protein synthesis, alkaline phosphatase activity, cel apoptosis, and cel migration in horizontal and vertical direction were observed.RESULTS AND CONCLUSION: The osteoblasts proliferation in the initial extract and secondary extract groups was better than that in the α-MEM medium group (P < 0.05). The osteoblasts proliferation in the initial extract group was better than that in the secondary extract group (P < 0.05). The total protein content of osteoblasts in the initial extract group was higher than that in the secondary extract and α-MEM medium group (P < 0.05). There were no significant differences in the alkaline phosphatase activity, apoptosis rate, horizontal migration distance of osteoblast and transmembrane cell number in Transwell between these three groups. These results demonstrate that borosilicate bioglass has good biocompatibility and has a certain benign regulatory role in osteoblast proliferation.