Objective To investigate the effects of genistein on the mRNA expressions of collagen (Col), matrix metalloproteinase (MMP ) and tissue inhibitor of matrix metalloproteinase (TIMP) in human embryonic skin fibroblasts (CCC-ESF-1).MethodsThe cultured CCC-ESF-1cells were divided into a black control group, an estradiol group and genistein groups of different doses. The mRNA expressions of ColⅠ, ColⅢ , MMP-1, TIMP-1 and TIMP-2 were detected by RT-PCR.Results Compared with the black group, estradiol and medium dose of genistein (0.451 ± 0.037, 0.446 ± 0.047vs.0.385 ± 0.061, allP<0.05) could promote the proliferation of the CCC-ESF-1 cells, estradiol and medium dose of genistein could up-regulate the mRNA expressions of ColⅠ (0.960 ± 0.012, 0.929 ± 0.015vs.0.812 ± 0.014, allP<0.01), ColⅢ (0.892 ± 0.009, 0.824 ± 0.022vs.0.768 ± 0.025, allP<0.01), TIMP-1 (0.841 ± 0.023, 0.838 ± 0.053vs.0.751 ± 0.027, allP<0.01) and TIMP-2 (0.456 ± 0.017, 0.448 ± 0.036vs.0.381 ± 0.029, allP<0.01), and down-regulate MMP-1 mRNAexpression (0.398 ± 0.043, 0.402 ± 0.044vs.0.525 ± 0.006, allP<0.01).Conclusions Genistein could promote the proliferation of the CCC-ESF-1 cells, and that may be related with up-regulating the mRNA expressions of ColⅠ, ColⅢ , MMP-1, TIMP-1and down-regulating MMP-1 mRNA expression.

Objective To explore the mechanism and regulative function of betulin on anti-aging genes related of normol human dermal fibroblasts (ESF-1), and to reveal the repairing mechanism of betulin on wrinkles. Methods The cultured cell in vitro were divided into the control group, the estradiol group and the betulin high, medium and low dose groups. Competence of ESF-1 cells was detected by MTT. Expression of mRNA of collagen type I and Ⅲ (ColⅠand Col Ⅲ), tissue inhibitors of matrix metalloproteinase 1 and 2 (TIMP-1 and TIMP-2), matrix metalloproteinase 1 (MMP-1) was detected by RT-PCR. Results Compared with the control group, the proliferation of ESF-1 cells (0.455 ± 0.037, 0.445 ± 0.040 vs. 0.385 ± 0.601) in the estradiol group and the medial-dose betulin group were increased (P<0.05). Compared with the control group, the expression of mRNA of ColⅠ (0.960 ± 0.012, 0.929 ± 0.015 vs. 0.842 ± 0.014), Col Ⅲ (0.892 ± 0.009, 0.824 ± 0.022 vs. 0.768 ± 0.025), TIMP-1 (0.938 ± 0.026, 0.878 ± 0.035 vs. 0.796 ± 0.022), TIMP-2 (0.557 ± 0.025, 0.506 ± 0.036 vs. 0.436 ± 0.063) in the estradiol group and the medial-dose betulin group were increased (all P<0.01). Conclusion Betulin based on increasing the competence of ESF-1 cells, promoting collagen synthesis and the expression level of related-proteinase is to suspend the development of wrinkles.

Objective To study the pinoresinol diglucoside (PDG) on gene regulation role of ESF-1 cells in collagen secretion, to reveal PDG repair mechanisms on scalded skin.Methods The cells cultured in vitro were divided into the control group, the estradiol group and the three different PDG doses groups. The concentration of the high, medium and low dose groups were 100, 10, 1μmol/L, and that of estradiol group were 10-3μmol/L. The activity of proliferation was detected by MTT. Then collagen type I (Col I), collagen typeⅢ (ColⅢ), tissue inhibitors of metalloproteinase 1 (TIMP-1), tissue inhibitors of metalloproteinase 2 (TIMP-2) and matrix metalloproteinase 1 (MMP-1) expression levels of mRNA after administration of cells were detected by RT-PCR.Results Compared with the control group, the proliferation of ESF-1 cells (0.559 ± 0.027, 0.552 ± 0.034vs. 0.489 ± 0.027,P<0.05) in the estradiol and medium-dose PDG was significantly higher. The expression level of mRNA of ColⅠ(0.958 ± 0.021, 0.929 ± 0.031, 0.916 ± 0.015vs. 0.844 ± 0.022), ColⅢ (0.783 ± 0.038, 0.918 ± 0.021, 0.855 ± 0.017vs. 0.678 ± 0.024), TIMP-1 (0.939 ± 0.025, 0.889 ± 0.036, 0.853 ±  0.015 vs. 0.780 ± 0.023), TIMP-2 (0.507 ± 0.024, 0.655 ± 0.037, 0.572 ± 0.025vs. 0.405 ± 0.062) in the estradiol, low-, medium-dose PDG groups were significantly higher than those in the control group (P<0.05 or P<0.01). Besides, the MMP-1 (0.343 ± 0.038, 0.407 ± 0.046, 0.435 ± 0.037vs.0.519 ± 0.041) mRNA expression level in the middle and low dose PDG groups significantly decrease (P<0.05 orP<0.01). Conclusions The PDG could enhance the activity of ESF-1 cell proliferation, increase the expression of related collagen and tissue inhibitor of metalloproteinases and inhibit that of matrix metalloproteinases to repair scalded skin.

Objective To study the anti-rheumatoid arthritis mechanism of Ermiao powder by high-throughput urine metabolomics.Methods The rats were randomly divided into control group,model group and administration group with 8 rats in each group.The rat model of rheumatoid arthritis was established by intradermal injection of complete Freund's adjuvant.Rats in control group were given Ermiao power solution 0.108 g/ml by gavage.The degree of joint swelling in rats was observed and scored.On this basis,metabolic data of rat urine samples were collected for metabolomic analysis.Unsupervised and supervised pattern recognition technology was used to analyze the high-throughput biological information data and reduce the dimension.Metabolic information related to rheumatoid arthritis was screened and focused to clarify the pathogenesis of rheumatoid arthritis and the therapeutic mechanism of Ermiao power.Results Compared with the model group,the swelling degree of the foot (1.93 ± 0.11 vs.2.36 ± 0.19) in Ermiao power group significantly decreased (P＜0.01).Metabolic profiles showed that the metabolic distribution of healthy rats was significantly separated from that of model rats,and the treatment group was in the middle of the two groups.From the macro-metabolic point of view,the metabolism of model rats changed dramatically.The Ermiao power had a good intervention effect on rheumatoid arthritis.Thirteen biomarkers related to rheumatoid arthritis were identified by database matching,including linolenic acid,arachidonic acid,5,6-EET,alpha-lactose,sucrose,trehalose,prostaglandins and leukotriene C4.It involved linoleic acid metabolism,arachidonic acid metabolism,starch and galactose metabolism and sphingolipid metabolism.Conclusions The Ermiao power has significant therapeutic effect on rheumatoid arthritis rats.Regulation of the linoleic acid metabolism,arachidonic acid metabolism,starch and galactose metabolism may be the mechanism of its treatment of rheumatoid arthritis.

OBJECTIVETo study the radiobiological characteristics of a HepG2 cell line with mitochondrial DNA (mtDNA) deletion.

METHODSHepG2 cells were cultured in a medium containing ethidium bromide, acetylformic acid and uracil. The HepG2 cell line with mtDNA deletion (ρ(0)HepG2 cells) were acquired after 30 subcultures by limited dilution cloning. The cell survival was then observed in the absence of acetylformic acid and uracil, and the total mtDNA deletion in the cells was confirmed by PCR. The radiosensitivity of HepG2 and ρ(0)HepG2 cells was evaluated by exposure to gradient doses of 6 MV X ray irradiation. The cell apoptosis was assessed following a 2 Gy X-ray exposure with Hochest33342 staining, and the invasiveness of ρ(0)HepG2 cells was measured by Transwell assay.

RESULTSHepG2 cells could survive 30 subcultures in the presence of ethidium bromide, and massive cell death occurred after removal of acetylformic acid and uracil from the medium. PCR confirmed total mtDNA deletion from ρ(0)HepG2 cells, whose α/β value was significantly lower than that of HepG2 cells. ρ(0)Hep-G2 cells showed an obviously lowered cell apoptosis rate following X-ray exposure with enhanced cell invasiveness.

CONCLUSIONHepG2 cells can be induced by ethidium bromide into ρ(0)HepG2 cells with an increased radiation resistance, anti-apoptosis ability and cell invasiveness.

Apoptosis ; Culture Media ; chemistry ; DNA, Mitochondrial ; genetics ; Ethidium ; chemistry ; Hep G2 Cells ; radiation effects ; Humans ; Radiation Tolerance ; genetics ; Sequence Deletion ; X-Rays

Objective:To study the effect of baicalein on the expression of glutamate receptor related protein in PC12 cells injured by Aβ 25-35. Methods:PC12 cells were divided into control group, model group, estradiol group and baicalein group with different concentrations. The survival condition of PC12 cells in each group were detected by thiazole blue (MTT). PC12 cells were divided into control group, model group, estradiol group and baicalein group. The control group and model group were cultured with DMEM medium, and the estradiol group was added with 1×10 -3 μmol/L estradiol DMEM medium, baicalein group was added with 1 μmol/L baicalein DMEM medium. After 2 hours of intervention, 20 μmol/L Aβ 25-35 was added to the model group, estradiol group and baicalein group with induced PC12 cell injury. After 22 hours of intervention, flow cytometry was used to detect the apoptosis of PC12 cells. The expression of estrogen receptor β (ER β), phosphorylated c-Jun N-terminal kinase (p-JNK/JNK) and ionic receptor N-methyl-D-aspartate receptor 1 (NMDAR1), glutamate receptor 2 (GluR2) and calcium/calmodulin dependent protein kinase Ⅱ (CaMK Ⅱ) were detected by Western blot. Results:Compared with model group, 1 μmol/L baicalein significantly increased the proliferation rate [(95.80±2.47)% vs. (64.34±3.84)%]. The apoptosis rate of PC12 cells[(7.83±0.67)% vs. (12.84±0.91)%] was significantly decreased in baicalein group ( P<0.01). The expression of NMDAR1 (0.582±0.012 vs. 0.352±0.012), GluR2(0.538±0.017 vs. 0.355±0.006), ER β (0.362±0.015 vs. 0.262±0.018) in baicalein group were significantly increased ( P<0.01), the expression of p-JNK/JNK (0.476±0.013 vs. 0.752±0.014) and CaMK Ⅱ(0.499±0.019 vs. 0.670±0.016) in baicalein group were significantly decreased ( P<0.01). Conclusions:Baicalein has a protective effect on PC12 cells injured by Aβ 25-35. Its mechanism may be related to the inhibition of p-JNK/JNK activity by activating ERβ and regulating the expression of glutamate receptor related protein.

Objective To study the prevalence of cigarettes and e-cigarettes, and to analyze its influencing factors among high school students in Wenzhou. Methods:A self-administered questionnaire survey was conducted among a total of 3 785students from senior high schools and vocational high schools in Wenzhou from September to November in 2020, who were selected by stratified cluster sampling. Their demographic information and the use of tobacco [including the use of cigarettes, e-cigarettes (currently smoking and smoking attempt), and the dual use of them] were asked in the questionnaire of “Global Youth Tobacco Survey”(GYTS). Proportion and rate were used to describe their demographic characteristics and smoking prevalence. Chi-square test was used to compare the difference in smoking prevalence of the high school students with selected characteristics. Multinomial logistic regression was used to analyze the influencing factors of tobacco use among the participants.Results:Among the 3 785 questionnaires, 3 740 were completed, and effective rate was 98.80%. The smoking attempt rate, current smoking rate, e-cigarette attempt rate, e-cigarette using rate, and dual using rate of the high school students in Wenzhou were 11.79% (441/3 740), 2.57% (96/3 740), 10.00% (374/3 740), 2.86% (107/3 740), 0.99% (37/3 740), respectively. Male students, vocational high school students, the students whose parents smoking showed higher smoking attempt rate, current smoking rate, e-cigarette attempt rate than female student, senior high school students and those parents who don′t smoke, respectively (all P<0.05). Best friend smoking [6.076 (2.872-12.855); 4.630 (2.656-8.071)], vocational high school [3.226 (1.877-5.546); 2.238 (1.432-3.496)] were risk factors for e-cigarette and the cigarettes use, belief that whether smoking or not does not make any difference in social activities [0.160 (0.077-0.331); 0.193 (0.096-0.387)], or smoking in social activities makes people more uncomfortable [0.037 (0.018-0.077); 0.155 (0.083-0.288)] were protective factors of e-cigarette and the cigarettes use. Additionally, passive smoking in past week [2.194 (1.129-4.262)], free tobacco products received [2.836 (1.018-7.905)], seeing smoking in campus [2.072 (1.111-3.863)] were all risk factors, while belief that smoking makes young people less attractive [0.280 (0.116-0.676)], not having been to tobacco retail outlets and unexposed to tobacco advertising or promotion [0.241 (0.123-0.473)] can protect senior middle school student away from cigarette. As to e-cigarette, boys were the risk factors [1.831 (1.100-3.050)]; as opposed to being a risk factor for cigarette use [1.942 (1.084-3.478)], rural area was a protective factor for e-cigarette use [0.639 (0.410-0.994)] among high school students in Wenzhou. Conclusions:High school students have relatively low rates of smoking cigarettes and e-cigarette in Wenzhou. Vocational students, best friend smoking are risk factors of e-cigarette and the cigarettes, beliefs that whether smoking or not does not make any difference or smoking makes people more uncomfortable in social activities are protective factors.

Objective To conduct genetic diagnosis and prenatal diagnosis for a haemophilia B family with multi-nucleotides deletion mutation of F9 gene.Methods This is a genetic analysis.Whole exon mutation of the F9 gene was analyzed by PCR and Sanger sequencing for seven patients with the family of hemophilia B who consulted doctors in Henan Province People′s Hospital in April 2013.Suspected mutation was verified among non-hemophilia B members of the family and 100 healthy controls to rule out genetic polymorphism of the F9 gene.The above-mentioned detection results of hemophilia B gene , the pathogenic mutation of F9 gene in the family was clarified , and prenatal diagnosis was conducted for the female carriers in the family.It is recommended that the fetal gene detection should be conducted in amniotic fluid in the mid-term pregnancy of the female carriers of hemophilia , and then they can be informed of the non-hemophilia B fetus by the results of the gene detection .Results PCR and sequencing analysis has identified a deletion mutation of F9 gene c.185_188delGAGA[p.Glu62Asnfs?41]in seven hemophilia B patients.This mutation induced F9 gene frame shift mutation which led to early termination of F9 gene translation because there was a termination codon TAA at the 41th codon after the mutation site.The same mutation was not found among the non-hemophilia B members of the family and the 100 healthy controls. There were eight female carriers and nine female non-carriers in the family.Upon prenatal diagnosis , the Y chromosome sex-determining gene ( SRY ) in amniotic fluid was positive and no deletion mutation was observed in the F9 gene c.185_188.Conclusion The pathogenic mutation of F9 gene in the family was identified , which was helpful for prenatal diagnosis in female carriers .

Objective: To explore the mechanism of Shengxian decoction in the treatment of heart failure by using metabolomic technology. Methods: Rats were randomly divided into the control group, model group and administration group according to body weight, with 10 rats in each group. Rats in model group and administration group were induced by intraperitoneal injection of adriamycin to duplicate rat heart failure model. The rats in the treatment group were given Shengxian decoction 3.83 g/kg, while those in the control group and model group were given distilled water of equal volume once a day for 4 weeks. The levels of CK, AST, LDH and MDA in serum of rats were detected by ultraviolet spectrophotometer, and the metabolite profiles were collected by high resolution tandem mass spectrometry. The data were analyzed by principal component analysis and partial least squares discriminant analysis. Metabolic pathways were obtained by pathway enrichment analysis, focusing on key metabolic enzymes and metabolic pathways. Results: Compared with the model group, the serum CK (1 015.44 ± 201.49 U/L vs. 1 301.89 ± 311.54 U/L), AST (210.59 ± 80.34 U/L vs. 421.56 ± 120.32 U/L), LDH (1 211.64 ± 416.61 U/L vs. 601.58 ± 311.74 U/L) in the administration group significantly decreased (P<0.05), and MDA (209.21 ± 151.15 nmol/ml vs. 1 251.15 ± 110.64 nmol/ml) levels significantly increased (P<0.05). The metabolic distribution of rats in the control group was significantly separated from that in the model group, and the administration group was between the two groups. After dimension reduction, blood biomarkers were obtained by partial least squares discriminant analysis, including citric acid, succinic acid, malic acid, arachidonic acid, canine uric acid, serine, sphingosine, Cer (d18:0/14:0), SM (d18:1/22:0), SM [d18:0/18:1 (11Z)], SM (d18:0/16:1). Metabo Analyst 4.0 analysis showed abnormal metabolism in heart failure rats, which mainly involved arachidonic acid metabolism, glycine, serine and threonine metabolism, sphingolipid metabolism, citric acid metabolism and aminoacyl-tRNA biosynthesis. Conclusions The Shengxian decoction has a good therapeutic effect on heart failure rats. Regulation of arachidonic acid metabolism, glycine, serine and threonine metabolism, sphingolipid metabolism, citric acid metabolism and aminoacyl-tRNA biosynthesis may be the key mechanisms for its treatment of heart failure.

Objective:To investigate the effects of down-regulation of FABP5 (fatty acid binding protein 5) on radiation damage of skin cells, and explore underlying mechanism.Methods:A lentiviral vector with down-regulated FABP5 was constructed to infect human immortalized keratinocytes (HaCaT) cells, and the transfection efficiency was examined. The HaCaT cells were divided into blank control group, FABP5 down-regulation group (FABP5), radiation group (IR), and FABP5 down-regulation combined with radiation group (FABP5+ IR). After 6 MV X-ray radiation, cell proliferation viability was measured by CCK-8 assay, cell migration was detected by scratch assay, apoptosis was analyzed by flow cytometry, radiosensitivity was evaluated by cloning formation assay, and the cellular protein expressions of PARP1, γ-H2AX, AKT and p-AKT were detected by Western blot.Results:FABP5 was successfully knocked-down in both RNA level ( t=25.14, P<0.05) and protein level ( t=20.06, P<0.05). The down-regulation of FABP5 decreased the abilities of cells proliferation ( t=3.55, 5.88, 3.18, P<0.05) and migration ( t=15.44, P<0.05), but increased cell resistance to irradiation with a radiosensitization ratio of 0.782. The apoptosis rate of FABP5+ IR group was significantly lower than IR group (22.05±6.71)% vs. (9.82±1.45)%, t=3.08, P<0.05. The protein levels of PARP1 and γ-H2AX in FABP5+ IR group were also lower than those in the IR group 0.04±0.04, 0.11±0.06, 0.26±0.11, 0.22±0.07, 0.21±0.10, 0.52±0.22, 0.57±0.06, 0.43±0.02( t=2.83, 3.07, 4.50, 5.33, P<0.05), while the protein level of p-Akt in FABP5+ IR group was higher than that in IR group ( t=-16.24—3.02, P<0.05). Conclusions:Down-regulation of FABP5 inhibited cell proliferation and migration, increased radioresistance, and reduced radiation-induced apoptosis and DNA damage of skin cells probably through PI3K/AKT signaling pathway.