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Objective To evaluate the effect of hydrogen peroxide (H2O2) on a senescence marker protein-30 (SMP30) and an autophagy-related protein microtubule-associated protein 1 light chain 3 type Ⅱ (LC3-Ⅱ) in normal human skin fibroblasts (NHSFs).Methods NHSFs were isolated from the foreskin of children,and subjected to culture in vitro.The second-to fourth-passage NHSFs were treated with 150 μmol/L H2O2 for 2 hours to establish a model for cellular senescence,while un-treated NHSFs served as control group.Senescence-associated β-galactosidase (SA-β-gal) staining was performed to determine the percentage of senescent cells,indirect immunofluorescence assay to determine the expression of the autophagy-related protein LC3,reverse transcription PCR (RT-PCR) to measure the mRNA expression of SMP30,and Western blot analysis to measure the protein expression of SMP30 and LC3.Results The percentage of senescent (SA-β-gal-positive) cells was significantly higher in the H2O2 group than in the control group (41.70％ ± 2.95％ vs.3.03％ ± 0.25％,t =22.59,P ＜ 0.05).Indirect immunofluorescence assay showed that the percentage of LC3-positive cells was significantly lower in the H2O2 group than in the control group (12.60％ ± 1.57％ vs.23.67％ ± 3.04％,t =5.61,P ＜ 0.05).As Western blot analysis showed,no significant difference in the expression of LC3-Ⅰ (LC3-Ⅰ/glyceraldehyde-3-phosphate dehydrogenase [GAPDH] ratio) was observed between the H2O2 group and control group (0.40 ± 0.02 vs.0.41 ± 0.04,P ＞ 0.05),while the H2O2 group showed significantly lower expression of LC3-Ⅱ (LC3-Ⅱ/GAPDH ratio:0.20 ± 0.02 vs.0.80 ± 0.03,t =29.69,P ＜ 0.05) and lower LC3-Ⅱ/LC3-Ⅰ ratio (0.51 ± 0.03 vs.1.98 ± 0.23,t =10.967,P ＜ 0.05) compared with the control group.Moreover,the mRNA and protein expression of SMP30 (SMP30/GAPDH ratio) was significantly lower in the H2O2 group than in the control group (mRNA:0.16 ± 0.01 vs.0.35 ± 0.01;protein:0.27 ± 0.02 vs.0.63 ± 0.02,both P ＜ 0.05).Conclusion H2O2 can decrease the expression of SMP30 and LC3-Ⅱ in NHSFs,and accelerate the senescence of NHSFs.
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Objective To observe the therapeutic effect of Shenluo'an Decoction(SD) for the treatment of obese patients with early diabetic nephropathy and to explore the possible mechanism.Methods A total of 68 obese patients with early diabetic nephropathy were randomly assigned into control group and treatment group,34 cases in each group.Both groups were given conventional western medicine treatment including lifestyle regulation,dietary control,and medicine for lowering blood pressure,blood glucose and blood lipids.Additionally,the control group was given oral use of Irbesartan,and the treatment group was given oral use of SD combined with Irbesartan.Before and after treatment,we observed the traditional Chinese medical syndrome scores,body mass index(BMI),blood levels of cystatin C (Cys C),β2-microglobumin(β2-MG),homocysteine (Hcy) and fasting blood glucose (FBG),and urine parameters of urinary monocyte chemoattractant protein-1 / urinary creatinine (UMCP-1/UCR),urinary factor of regulated on activation in normal T cell expressed and secreted / urinary creatinine (URANTES/UCR),urinary albumin-to-creatinine ratio (UACR) and urinary albumin excretion ratio (UAER) before treatment and after treatment for 16 weeks.After treatment,the clinical efficacy and safety were also evaluated.Results (1) During the treatment,5 cases were dropped out,and a total of 63 cases (32 from the treatment group and 31 from the control group) completed the experiment.(2) The total effective rate of the treatment group(90.6％) was significantly higher than that of the control group(67.7％),the difference being significant(P ＜ 0.05).(3)BMI of the two groups after treatment was significantly lower than that before treatment (P ＜ 0.05).(4) After treatment,the traditional Chinese medical syndrome scores,blood parameters (Cys C,β2-MG,Hcy,FBG) and urine parameters (UMCP-1/UCR,URANTES/UCR,UACR,UAER) in the treatment group were decreased as compared with those before treatment and those in the control group after treatment(P ＜0.05).(5) A positive correlation was present among the parameters of UMCP-1/UCR,URANTES/UCR,UACR and UAER.(6) No abnormal changes took place in the blood and stool routine examination,hepatic function,or electrocardiography of the 63 cases.Conclusion SD combined with western medicine exerts certain therapeutic effect for obese patients with early diabetic nephropathy,and is effective on relieving clinical symptoms anddecreasing BMI,Cys C,β2-MG,UACR,UAER,UMCP-1 and URANTES.Its mechanism is probably related with the inhibition of chronic renal inflammation.
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Objective To investigate the effect of highly expressed β-catenin on the proliferative activity of and expressions of two apoptosis-related genes Bcl-2 and Bax by human skin fibroblasts induced by hydrogen peroxide (H2O2).Methods Normal human skin fibroblasts (HSFs) from child foreskin were divided into three groups:empty vector group transfected with the empty vector pcDNA3.1,H2O2 group transfected with the empty vector pcDNA3.1 followed by treatment with H2O2 (150 μ mol/L) for 2 hours,β-catenin group transfected with pcDNA3.1-β-catenin followed by treatment with H2O2 (150 μ mol/L) for 2 hours.Subsequently,methyl thiazolyl tetrazolium (MTT) assay was conducted to estimate proliferative activity of fibroblasts,flow cytometry to detect cell apoptosis,and reverse transcription (RT)-PCR and Western blot were performed to measure the mRNA and protein expressions of Bcl-2 and Bax respectively.The relative expression levels of genes were expressed as the ratios between the targets and GAPDH.Results Significant differences were found between the empty vector group,H2O2 group and β-catenin group in cellular proliferative activity (expressed as absorbance value at 570 nm:0.792 ± 0.012 vs.0.462 ± 0.012 vs.0.521 ± 0.015,P＜ 0.01) and apoptosis rate (3.407％ ± 0.217％ vs.24.555％ ± 1.793％ vs.15.360％ ± 0.755％,P＜ 0.01).Both mRNA and protein expression levels of Bcl-2 were significantly lower in the H2O2 group (0.333 ± 0.003 and 0.336 ± 0.004 respectively) than in the empty vector group (0.507 ± 0.013 and 0.514 ± 0.021,respectively,both P ＜ 0.01) and β-catenin group (0.404 ± 0.006 and 0.411 ± 0.005,respectively,both P ＜ 0.01).Increased expression levels of Bax mRNA and protein were observed in the H2O2 group compared with the empty vector group and β-catenin group (mRNA:0.451 ± 0.002 vs.0.303 ± 0.005 and 0.339 ± 0.012,protein:0.460 ± 0.008 vs.0.320 ± 0.013 and 0.346 ± 0.013,all P＜ 0.01).Conclusion High expression of β-catenin can raise proliferative activity of aging HSFs.
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Objective To investigate clinical curative effect of partial foraminotomy after C4.5 ossification of posterior longitudinal ligament (OPLL) on preventing C5 nerve root palsy.Methods A total of 155 patients with OPLL were treated with expansion open-door laminoplasty (EOLP) between January 2008 and October 2012,including 75 male patients and 80 female patients,with average age of 54.5 (ranging from 38 to 75).Among them,90 cases took EOLP (open team),and 65 cases took EOLP and C4.5 partial foraminotomy (incision team).Clinical curative effect and imaging evaluation were used in 8 weeks and 12 months after operation.JOA scores,JOA scores improvement rate,C5 nerve root palsy occurrence rate,neurological function of patients with C5 nerve root palsy and the prognosis,cervical curvature index,opening angular variation,backward drift distance of anterior margin and posterior margin of the spinal cord were evaluated after 8 weeks and 1 year after the surgery.Results The operation time was 90-140 min,with the average time of 110±14 min;mean bleeding volume was 160±45 ml (ranging from 100 to 250 ml);follow-up visit time was 15.4 months on average (ranging from 12 to 18 months).JOA score and improvement rate after 8 weeks and 1 year of the operation have no statistical significance.The C5 nerve root palsy occurrence rate in open team was 8.89％ (8/90),while that in incision team was 4.62％ (3/65).The difference of two groups has statistical significance.C5 nerve root palsy all happened in laminar opening side.Comparison of cervical curvature index of two groups before the operation and after 8 weeks of the operation and inter-group comparison have no statistical significance.The difference between laminar opening angle after 1 year of the operation and laminar opening angle after 8 weeks has no statistical significance.The difference of backward drift distance of anterior margin of the spinal cord at different time after the operation and inter-group difference have no statistical significance.The difference in backward drift distance of posterior margin of the spinal cord between 1 year after the operation and 8 weeks after the operation has no statistical significance.Conclusion Treating OPLL with EOLP and C4.5 partial foraminotomy can reduce occurrence rate of C5 nerve root palsy,but has no significant influence on JOA score improvement rate.
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Objective To compare the therapeutic efficacies of fire-needle acupuncture and Halometasone cream in treating sub-acute and chronic eczema. Method Totally 114 patients were divided into a treatment group and a control group. The treatment group was intervened by using fire-needle acupuncture, and the control group was by Halometasone cream. Itchy score and Eczema Area and Severity Index (EASI) were used for comparison before and after intervention. Result There were significant differences in comparing the markedly-effective rate and total effective rate between the fire-needle acupuncture group and the Halometasone control group according to the itchy score (P＜0.01). There were no significant differences in comparing the markedly-effective rate and total effective rate between the two groups according to EASI (P>0.05). Conclusion Fire-needle acupuncture is superior to Halometasone cream for acute and chronic eczema in relieving itch, action time and effect-sustaining duration.
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Objective To observe the effect of high expression of β-catenin on senescent phenotypes in normal human skin fibroblasts (NHSFs) induced by hydrogen peroxide (H2O2).Methods Cultured NHSFs were classified into three groups: β-catenin + H2O2 group transfected with a recombinant plasmid pcDNA3.1-β-catenin and treated with H2O2 of 150 μ mol/L for two hours,H2O2 group transfected with the empty vector pcDNA3.1 and treated by H2O2 of 150 μmol/L for two hours,and vector group transfected with the empty vector pcDNA3.1 and receiving no treatment.Reverse transcription (RT)-PCR and Western blot were performed to quantify the mRNA and protein expressions of β-catenin in these cells,microscopy to observe the morphological changes of cells.The activity of senescence-associated β-galactosidase (SA-β-Gal) and superoxide dismutase (SOD) as well as the level of reactive oxygen species (ROS) were detected by using commercial kits.Data were processed with the software SPSS 13.0,and analysis of variance (ANOVA) was conducted for multiple group comparisons.Results The expression of β-catenin was significantly upregulated in NHSFs transfected with the recombinant plasmid pcDNA3.1-β-catenin.Both the mRNA and protein expression levels of β-catenin described as β-catenin/ glyceraldehyde-3-phosphate dehydrogenase (GAPDH) ratio were significantly lower in the H2O2 group compared with the vector group (0.2900 ± 0.0195 vs.0.5963 ± 0.0400,0.3130 ± 0.0171 vs.0.6190 ± 0.0090,both P ＜0.05),while the protein expression level of β-catenin was statistically higher in the β-catenin + H2O2 group than in the H2O2 group (0.7953 ± 0.0074 vs.0.3130 ± 0.0171,P ＜0.05).Significant differences were observed between the vector group,H2O2 group and β-catenin+ H2O2 group in the percentage of SA-β-gal-positive cells ((2.9667 ± 0.2517)％ vs.(37.70 ± 0.9539)％ vs.(29.330 ± 0.6359)％,P ＜0.05),ROS activity ((50.9963 ±9.2688)％ vs.(109.9190 ± 11.5215)％ vs.(75.1063 ± 3.0138)％,P ＜0.05),and SOD levels ((88.0856 ±3.9181) vs.(35.5585 ± 3.4438) vs.(61.7029 ± 3.1716) U/mg,P ＜0.05).Conclusion The overexpression of β-catenin can downr_egulate the activity of SA-β-Gal and ROS level,but enhance the activity of SOD.
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Objective To observe the changes of β-catenin expression in human skin fibroblasts (HSFs) after induced by oxidative stress, and to explore its possible roles in oxidative stress-induced premature senescence (SIPS) of HSFs. Methods Fibroblasts were isolated from the foreskin of a child and subjected to a primary culture. The fibroblasts of second to fourth passage were treated with various concentrations of H2O2 for 2 hours to establish an optimized model of stress-induced premature senescence, β-galactosidase assay kit was used to detect the activity of β-galactosidase in H2O2rinduced HSFs, RT-PCR and Western blot to measure the mRNA and protein expressions of β-catenin in control and senescent HSFs. Results Premature senescence of HSFs could be induced by the treatment with H2O2 of 150 μmol/L for 2 hours. The proportion of β-galactosidase-positive cells was (2.97 ± 0.25)% in control HSFs and (37.67 ± 1.53)% in senescent HSFs (P< 0.01). A significant increase was observed in the β-catenin/GAPDH protein ratio and β-catenin/GAPDH mRNA ratio in control HSFs compared with the senescent HSFs (0.62 ± 0.03 vs. 0.31 ± 0.01, t = 14.97, P < 0.01; 0.59 ± 0.04 vs. 0.29 ± 0.30, t = 10.06, P < 0.01). Conclusions The two-hour treatment with H2O2 of 150 μmol/L could induce the premature senescence of HSFs, and there is a notable decrease in the expression of β-catenin in prematurely senescent HSFs induced by oxidative stress, implying that β-catenin is an important target gene for the regulation of skin aging.
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Objective To observe the influence of Shenkang injection with Irbesartan on urinary albumin excretion rate(UAER)in 24 hours,serum lipid and blood rheology in early diabetic nephropathy.Methods One hundred and fifty normotensive early diabetic nephmpathy were randomly divided into control group,Shenkang injection group and Shenkang injection with Irbesartan group.Three groups were treated with diabetic education,dietary regimen,suitable physical activity and oral anti-hyperglycemic agents till their fasting blood glucose(FBG)was less than 7.0 mmol/L and postprandial blood glucose(PBG)was less than 10.0 mmol/L.The control group was treated with oral anti-hyperglycemic agents or/and insulin.Shenkang injection group was additionally treated with Shenkang solution.Shenkang solution was mixed by Shenkang injection 100 ml,5% glucose 250 ml and insulin 2-3 U.Shenkang solution was intravenously guttaed once a day.Shenkang injection with Irbesartan group was additionally treated with Irbesartan 0.15 g,once a day,taken orally.A course was taken for 3 weeks.The levels of UAER,serum lipid and blood rheology were detected immediately and 9 weeks after treatment.Results In the Shenkang injection group,compared with those before treatment,them were significant difference in UAER (87.44 ±10.06)μg/min vs.(116.55 ± 33.42)μg/min),serum lipid( (4.22 ± 0.70)mmol/L vs.(4.88 ± 0.69 )mmol/L)and blood rheology( (5.77 ±0.53)mPa·s vs.(7.38 ± 0.41)mPa·s)(P ＜ 0.05).In the Shenkang injection with Irbesartan group,compared to those before treatment,there were also significant difference in UAER ( (61.90 ±28.02)μg/min vs.(123.37 ± 29.98)μg/min),serum lipid( (4.00 ± 0.14)mmol/L vs.(4.90 ± 0.12)mmol/L)and blood rheology( (5.11 ±0.41)mPa·s vs.(7.27 ± 0.44)mpa·s)(P ＜ 0.05).Whearas in the control group,no significant difference were found compared with those before treatment.Furthermore,the improvements in the Shenkang injection with Irbesartan group were better than those in the Shenkang injection group(P ＜ 0.05).Conclusions Shenkang injection can reduce UAER,decrease serum lipid and improve renal blood rheology.Iteffectively prevents diabetic nephropathy in earlier period and maybe have synergy with Irbesartan.
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In this case-control study, the relationship between M196R (676 T-->G) variant in exon 6 of tumor necrosis factor receptor type 2 ( TNFR2 ) gene and genetic susceptibility of acne vulgaris in Han Chinese was investigated. A total of 93 acne vulgaris patients and 90 healthy subjects from Han Chinese ethnic group were enrolled in this study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique was adopted to analyze the single nucleotide polymorphisms (SNPs) of TNFR2 M196R gene, and to examine the association between acne vulgaris and the polymorphisms in TNFR2 M196R gene. The relationship between different genotypes and the susceptibility of acne vulgaris was analyzed. The results showed that there was significant difference in the frequency of the genotype M/R+R/R in the TNFR2 M196R genetic polymorphisms between acne vulgaris patients and healthy controls (chi(2)=4.343; P=0.037; OR=1.899; 95% CI: 1.036-3.445); and there was significant difference in the allele (R) frequency between acne vulgaris patients and healthy controls (chi(2)=5.588; P=0.018; OR=1.838; 95% CI: 1.105-3.057). It was concluded that the high frequency of 196R allele in the functional M196R polymorphism of TNFR2 is a risk factor for acne vulgaris in Han Chinese.