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Objective To investigate the effects and mechanisms of carbon monoxide-releasing molecule-2 (CORM-2) on LPS induced barrier injure of Caco-2 cells.Methods The model of Caco-2 monolayer cells damage induced by LPS was established by using 50 μg/ml LPS for 24 hours.After preconditioning with different concentrations (10 μmol/L,50 μmol/L,and 100 μ mol/L) of CORM-2 for 1 hour,the cultured well-grown Caco-2 monolayer cells were stimulated with 50 μ g/ml lipopolysaccharides (LPS) for 24 hour.The 100 μmol/L CORM-2 was put into 37℃ and 5％ CO2 incubator for 18 hours until the CO has been fully released,and it became an inactive CORM-2(iCORM-2).The cultured Caco-2 monolayer cells were divided into six groups:the control group,the LPS group,the LC1(10 μmol /L CORM-2 preconditioning) group,the LC2(50 μmol/L CORM-2 preconditioning) group,the LC3(100 μmol/L CORM-2 preconditioning) group,and the LC4 (iCORM-2 preconditioning) group.The apoptosis rates of different groups of Caco-2 monolayer cells were detected using flow cytometry.Cytokines (TNF-α,IL-1β and HMGB-1) levels of different groups were detected using ELISA kits.The levels of tight junction proteins (occludin ZO-1,claudin-1 and claudin-4) of every group were detected using Western blotting with specific antibodies.The structural changes of tight junction proteins were visualized by immunofluorescence technique.Results Compared with control group,cell apoptosis rate and release of inflammatory cytokines such as TNF-α,IL-1β and HMGB-1 in LPS group were significantly higher,and the levels of tight junction proteins were apparently decreased (P＜0.05) in LPS group.Compared with LPS group,cell apoptosis rate and release of inflammatory cytokines such as TNF-α,IL-1β and HMGB-1 decreased,and the levels of tight junction proteins were attenuated obviously,P＜0.05,in CORM-2 preconditioning groups.And the higher the concentration of CORM-2,the more obvious the protective effects.Conclusions This study demonstrates that CORM-2,as one of exogenous CO-releasing molecules,has the capacity to protect the barrier damage of LPS-stimulated Caco-2 monolayer cells in a concentration dependent manner.The higher the concentration of CORM-2 was,the stronger the protective effects were.The protective effects of CORM-2 include reducing Caco-2 monolayer cells apoptosis rate,inhibiting inflammatory cytokines production and release,and restoring distribution and levels of tight junction proteins.
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Objective To observe the changes of telomere length and MMPs level in human fibroblasts induced by UVB, and to explore their roles on skin photoaging .Methods Human skin fibroblasts were extracted and cul-tured.The 5th fibroblasts were irradiated by UVB .The morphology of fibroblasts were microscoped , and the length of telomere and the mRNA expression of COL1a1 and hTERT were detected by RT-qPCR.The expression of MMP-3 and MMP-1 were detected by Western blot .Results The fibroblasts gradually became round , wrinkled and disorderly arranged after 30 mJ/cm2 UVB irradiation for 24 h.The mRNA level of COL1a1 and hTERT and the expression of MMP-3 and MMP-1 were significantly increased after UVB irradiation compared with control , and the length of telomere was shortened .Conclusions UVB may frigger the early process of photoaging by the morphologi-cal changes of human skin fibroblasts and increasing the expression of MMP-3 and MMP-1 .
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Objective To investigate the effect of extracellular histones (EH) on intestinal mucosal barrier function in mice and the correlation of EH with the pathogenesis of sepsis.Methods Twenty male C57BL/6 mice were assigned into experiment group (n =10) and control group (n =10) according to the random number table.Same dose (50 mg/kg) of EH and saline were administered through the caudal vein of mice in experiment and control groups respectively.Blood and intestinal samples in each group were collected 3 h after the administration.Morphology of intestinal mucosal tissue was detected by light scope and transmission electron microscope.Expressions of tight junction related proteins (ZO-1,Occludin and Claudin-1) were detected by western blot.Plasma levels of diamine oxidase (DAO) and intestinal fatty acid binding protein (I-FABP) were determined by ELISA method.Plasma level of endotoxin (ET) was determined by limulus test.Results Under transmission electron microscope,experiment group showed disorganized microvilli of intestinal epithelial cells with partially twisted,broken and lost,unclear tight junctions,and widened cellular space.Under light scope,experiment group showed substantial inflammatory cell infiltration in the intestinal wall,disorganized intestinal villi,edema and hemorrhage of mucosa and submucosa,and edematous goblet cells.Experimental versus control group showed significant reduction in levels of Claudin-1 (0.587 7 ±0.060 6 vs.0.677 2 ±0.038 3),Occludin (0.1277±0.0857vs.0.4306±0.0869) and ZO-1 (0.393 3±0.080 8 vs.0.812 8± 0.096 3) (P ＜ 0.05).Experimental versus control group showed significantly up-regulated plasma levels of DAO [(1.61 ±0.20) U/ml vs.(0.69 ± 0.15) U/ml],I-FABP [(548.5 ± 36.8) EU/ml vs.(178.8±26.9) EU/ml] andET [(0.182±0.076) EU/mlvs.(0.091 ±0.029) EU/ml](P＜0.05).Conclusion EH can obviously impair the integrity of intestinal mucosal barrier in mice and hence induce endotoxin translocation.
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Objective To establish a mouse model of biliogenic severe acute pancreatitis (SAP) by using a self-made device for retrograde injection of sodium taurocholate into common bile duct,and to investigate the improvement of the device on retrograde injection of sodium taurocholate into common bile duct and its safety.Methods Thirty-six adult male ICR mice were randomly divided into biliogenic SAP model group and sham group,with 18 mice in each group.A 40 U disposable insulin syringe,a 200 μL tips and a 25 μL micro-syringer were used as basic materials for making the mouse common bile duct injection device [National Utility Model Patent (ZL 2014 2 0694365.4)].In model group,3.5％ sodium taurocholate (1 mL/kg) was injected retrogradely into the common bile duct of mice,whilst in sham group,the mice underwent the injection of equal amount of normal saline instead.Six mice in each group were sacrificed at 6,24 and 48 hours after operation,and the abdominal aortic blood was collected.Serum amylase (AMY),alanine aminotransferase (ALT),creatine kinase-MB (CK-MB),serum creatinine (SCr),oxygenation index (PaO2/FiO2) as well as serum Ca2+ were.determined.Pathological change in pancreas was observed under conventional light microscopy after hematoxylin and eosiu (HE) staining,and the impairment was evaluated by a widely used score system.Results The injection device was easily placed into mouse common bile duct under macroscopic observation.Six hours after operation,the levels of serum AMY,ALT and SCr in model group were significantly higher than those in sham group,and peaked at 24 hours,and they slightly decreased at 48 hours,which were still significantly higher than those of the sham group [24-hour AMY (U/L):7 325 ± 1 154 vs.1 737 ± 197,24-hour ALT (U/L):176.0±5.0 vs.38.3 ± 2.0,24-hour SCr (tmol/L):46.3 ± 1.5 vs.17.8 ±0.6,all P ＜ 0.01].The level of CK-MB at 6 hours in the model group was significantly higher than that of the sham group,and peaked at 48 hours (U/L:749.8±42.2 vs.383.3±35.5 at 6 hours,3 340.1 ± 203.6 vs.704.6 ± 63.5 at 48 hours,both P ＜ 0.01).PaO2/FiO2 at 6 hours after the operation in model group was significantly lower than that of sham group,then it began to rise at the similar level in sham group at 48 hours [mmHg (1 mmHg =0.133 kPa):327.5±33.8 vs.424.8±31.0 at 6 hours,P ＜ 0.01;429.8 ±41.8 vs.464.7±43.3 at 48 hours,P ＞ 0.05].Ca2+ level in model group was continuously decreased after operation,and it was significantly lower than that of sham group at 48 hours (mmol/L:1.58 ± 0.14 vs.2.45 ± 0.21,P ＜ 0.01).The pancreatic edema was obvious after operation in sham group,with the observation time prolongation,the changes were gradually improved;pancreatic focal necrosis was found at 6 hours after operation in model group,and it was secondary aggravated,and pancreatic lobule structure disappearance and inflammatory cells extensive infiltration was found at 48 hours.Pathological score of the model group was significantly higher than that of sham group at each time point,and peaked at 48 hours (13.3 ±0.3 vs.3.0±0.1,P ＜ 0.01).Conclusion It is a highly efficient and low-cost way to induce biliogenic SAP in mice by retrograde injection of 3.5％ sodium deoxycholate into common bile duct via the self-made injection device,and the model conformed to the clinical characteristics of biliogenic SAP.
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Objective To explore the significant risk factors in patients with Tile C pelvic frac-tures.Methods We conducted a retrospective review of all patients with Tile C pelvic fractures in Nanjing First Hospital from January 2010 to December 2014.The data gathered on each patient in-cluded:age,sex,mechanism of injury,visiting time after injury,shock index,Injury severity scale (ISS),Revised trauma score (RTS),Glasgowcoma scale (GCS),lowest PaO2/FiO2 ,6 h lactate clearance rate,concomitant injures and interventious of Tile C pelvic fractures.The data were tested using Student’t-test,χ2 test and logistic regression method.Results The study include 139 consecutive patients.The total mortality was 29.5%.Among them,36 (25.9%)patients died within 48 hours after admission.Multivariate regression analysis showed that shock index (OR=2.591,95%CI 1.041-4.216), ISS (OR = 47.96,95%,CI 15.89-147.23 ),RTS (OR = 6.917,95% CI 1.147-13.862 ),GCS (OR =4.172,95%CI 2.962-6.268),lowest PaO2/FiO2 (OR= 117.016,95% CI 51.011-176.032),6 h lactate clearance rate (OR=2.785,95%CI 1.191-4.892),concurrent head (OR=6.302,95%CI 2.270-13.175)or chest (OR=12.233,95%CI 5.193-33.985)injures were associated with high morality of Tile C pelvic frac-ture (P <0.01).The performing digital subtraction angiography with intravascular embolization can cut the mortality (OR=0.887,95%CI 0.875-0.899).Conclusion In our study,high trauma score,serious shock, coma,PaO2/FiO2 decreased and 6 h lactate clearance rate decreased,combined with the head and chest inju-ry are the important reasons of mortality in patients with Tile C pelvic fracture.It’s vital to control shock actively,use trauma scale and emphasize multidisciplinary cooperation to reduce mortality in patients with Tile C pelvic fractures.
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Objective To compare the different characteristics of adaptive support ventilation (ASV) and synchronized intermittent mandatory ventilation-pressure support ventilation (SIMV-PSV) mode in weaning patients after general anesthesia.Methods One hundred and twenty-eight patients received general anesthesia,ending in odd and even numbers by hospital number divided into ASV group (single number,62 cases) and SIMV-PSV group (double number,66 cases).The propofol dosage,duration of mechanical ventilation,duration of intubation,ventilator alarms,ventilator settings manipulations and each stage of the blood gas analysis,hemodynamic,respiratory mechanics were recorded.Results One hundred and twenty-eight patients completed the extubation.The propofol dosage,duration of mechanical ventilation,duration of intubation in ASV group were significantly shorter than those in SIMV-PSV group [(1.13 ± 0.33)mg/kg vs.(1.28 ±0.49) mg/kg,(169.8±36.5) min vs.(201.9 ±37.3) min,(197.2 ±38.9) min vs.(239.5 ± 42.3) min,P ＜ 0.05].There was no statistically significant difference in various stages of heart rate,mean arterial pressure,central venous pressure,pH,arterial carbon dioxide partial pressure,oxygenation index between two groups (P ＞0.05).In the first and second stages,tidal volume in ASV group was significantly higher than that in SIMV-PSV group [(543.6 ± 72.3) ml vs.(489.5 ± 68.7) ml,(513.9 ± 65.7)ml vs.(462.8 ± 61.7) ml,P＜ 0.05],respiratory rate in ASV group was significantly lower than that in SIMV-PSV group [(13.2 ± 3.6) times/min vs.(17.2 ±4.1) times/min,(15.1 ± 3.1) times/min vs.(16.8 ± 3.7)times/min,P ＜ 0.05].In the first stage,the mean airway pressure and peak airway pressure in ASV group were significantly lower than those in SIMV-PSV group [(8.2 ± 1.7) cm H2O (1 cm H2O =0.098 kPa) vs.(12.3 ± 3.1) cm H2O,(16.2 ± 2.9) cm H2O vs.(21.2 ± 4.3) cm H2O,P ＜ 0.05].The pulmonary dynamic compliance in ASV group was better than that in SIMV-PSV group [(64.8 ± 12.3) ml/cm H2O vs.(52.6 ±13.5) ml/cm H2O,P ＜ 0.05].The ventilator alarms,ventilator settings manipulations in ASV group were significantly shorter than those in SIMV-PSV group [(2.3 ± 1.6) times vs.(5.1 ± 1.9) times,(0.8 ± 0.5) times vs.(1.6 ± 0.8) times,P ＜ 0.05].Conclusion ASV weaning mode is safe and effective,which could accelerate the extubation and simplify the manipulation.
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ObjectiveTo develop a new nested PCR to detect human papillomavirus (HPV) DNA in lesions of extragenital Bowen's disease.Methods DNA was extracted from the lesions of 41 patients with extragenital Bowen's disease and suhjected to the amplification of HPV by a nested PCR.Five primers,including CN1FR,CN2FR,CN3FR,CN4FR and CN5FR,were designed and used in the second round PCR.ResultsBased on the ClustalX analysis,69 HPV subtypes,including mucosal types,cutaneous types and epidermodysplasia verruciformis-associated types,could be amplified by using the 5 designed primers.The detection limit varied from 10-3 to 10-2 copies of HPV DNA for this PCR.Of the 41 lesional specimens,5 were positive for HPV DNA,including 3 cases of high-risk HPV types(2 cases of HPV 16,1 case of HPV 33) and 2 cases of cutaneous HPV types(1 case of HPV 27 and 1 case of HPV 76).ConclusionsThe improved nested PCR is highly sensitive and specific for the detection of HPV DNA in lesions of extragenital Bowen's disease.The development of extragenital Bowen's disease may be associated with the infection with high-risk mucosal HPV types in some patients.
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Objective To investigate the changes of IL-8 in peripheral blood of patients with pustular psoriasis after acitretin treatment.Methods Dual antibody sandwich enzyme-linked immunosorbent assay (ELISA)was performed to measure the IL-8 levels in the peripheral blood of 30 patients with pustular psoriasis and 30 patients with psoriasis vulgaris before and after treatment,and in 30 normal human controls.The relationship between the IL-8 level and disease severity was assessed for patients with pustular psoriasis.Results After acitretin treatment,the condition was improved greatly in patients with pustular psoriasis,together with a significant decrease in the level of IL-8 in the peripheral blood(57.07 ± 12.02 pg/ml vs.96.84 ± 14.68 pg/ml,P ＜ 0.05).Conclusion IL-8 may be involved in the therapeutic mechanism of acitretin in pustular psoriasis.
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Objective To investigate the effects of low-dose of hydrocortisone on circulating thymus-dependent lymphocyte (T lymphocyte) apoptosis in patients with septic shock. Method fifty-seven patients with septic shock admitted into ICU from January 2006 to January 2009 were prospectively randomized (random number) to treatment group and control group. Another 20 healthy volunteers and 18 patients with sepsis alone were included as external control groups.The patients of treatment group and control group were treated with low-dose of hydrocortisone and placebo,respectively. Samples of peripheral blood were taken from healthy volunteers and patients 0 hr,24 hrs,48 hrs,72 hrs and 168 hrs after onset of the disease to determine the circulating T lymphocyte apoptosis by using the assays of Annexin V and flow cytometry. Least significant difference t -test was used for multiple comparisons. Results The percentage of Annexin V-positive CD4+ T lymphocytes in the primary stage was (11.01 +4.52)% in septic shock patients, (4.41 + 1.45)% in healthy volunteers, and (7.87 + 3. 82)% in patients with sepsis alone. And in the initial setting, the percentage of Annexin V-positive CD4+ T lymphocytes in the septic shock patients was higher than that in healthy volunteers ( P < 0.05) and in patients with sepsis alone ( P < 0.05). The percentage of Annexin V-positive CD8 + T lymphocytes at the beginning was (11.33+19.62)% in septic shock patients, (9.62+8.32)% in healthy volunteers, and (13.09+ 15.84)% in patients with sepsis alone (P > 0.05 between three groups). The percentages of Annexin V-positive CD4+ T lymphocytes in control group after 24 his, 48 hrs and 72 hrs were(13.51+6.85)%, (19.39 + 6.63)% and (15.33+ 6.21)%, respectively. And the percentages of Annexin V-positive CD4+ T lymphocytes in treatment patients after 24 hrs, 48 hrs and 72 hrs were (17.4 + 7.21)%, (22.61 + 5.64)%, and (25.73 + 6.91)%, respectively. The percentage of Annexin V-positive CD4+ T lymphocytes in septic shock patients was higher than that in control groups ( P < 0.05). The percentages of Annexin V-positive CD8+ T lymphocytes in control group after 24 hrs, 48 hrs and 72 hrs were (11.49+ 11.73)%, (12.74+ 10.39)% and (13.28+ 16.6)%, respectively, and in the treatment group, those were (9.49 + 8.9)%, (15.32+18.17)% and (13.68+16.84)%, respectively (P >0.05 between two groups). In the meantime, the percentages of Annexin V-positive CDS'1' T lymphocytes in control group and in treatment group were (12.72+ 19.69)% and (13.88 + 13.28)%, respectively (P >0.05). Conclusions Low-dose of hydrocortisone could induce CD4+ T lymphocyte apoptosis and has no effects on CD8+ T lymphocyte apoptosis when it is used to treat septic shock.
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Objective To assess the relationship between human papillomavirus (HPV) and extrageni-tal Bowen's disease. Methods Regular PCR with consensus primers for LI region as well as mix primers and nested PCR were performed to detect the DNA of a broad range of cutaneous and mucosal HPV types in tissue samples from lesions of 41 patients with extragenital Bowen's disease and from normal skin of 48 human controls. Semiquantitative PCR and tyramide-based in situ hybridization (ISH) were also conducted to determine the load and localization of HPV DNA in HPV-positive samples. Results HPV DNA was detected in lesions from 5 (12%) of the 41 patients with extragenital Bowen's disease. Of the 5 HPV-positive patients, 3 carried mucosal HPV types (HPV16 in 2 cases, HPV 33 in 1 case) with a viral load of 101 to 103 copies, 2 cutaneous HPV types (HPV27 in 1 case and HPV76 in 1 case). As ISH showed, there was a generalized expression of mucosal HPV DNA in most tumor cell nuclei but not in peritumoral normal tissue, and no expression of cutaneous HPV DNA was observed in lesions. HPV DNA was detected in 1 (2.1%) control tissue sample, which proved to be epidermodysplasia verruciformis-associated HPV23. There was no significant difference in the detection rate of HPV DNA between the patients and controls. The viral load of cutaneous HPV types amounted to 10-2 to 10-3 copies in the 2 patients, which was similar to that of HPV 23 in the normal control. Conclusion Mucosal HPV types may be closely associated with the development of extragenital Bowen's disease.
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BACKGROUND:Study on antisense drug is still one of hotspots in the current field of biomedicine. Due to high-efficiency and specificity, antisense drug used for gene therapy has been paid more attention by many scholars.OBJECTIVE: To observe the effect of liposome-mediated keratin 14 (K14) antisense oligonucleotide on K14 gene and protein expressions as well as in vitro proliferation activities in human keratinocytes (KC).DESIGN: Single sample observation.SETTING: Department of Dermatology, Xijing Hospital, Fourth Military Medical University of Chinese PLA.MATERIALS: Human KC, K14 oligonucleotide gene fragments (modified with phosphrothioate, and above sequence was synthesized by Shanghai Shenggong Bioengineering Company). Reverse transcriptase and TaqDNA polymerase were purchased from Invitrogen Company, K14 monoclonal antibody was purchased from Antibody Company, and SABC kit was purchased from Boster Company. EPICS-PRO-FILE Ⅱ flow cytometer was purchased from Coulter Company (USA).METHODS: Human epidermal KCs were primarily cultured, and their 3rd to 10th generations were used for the experiment.Artificially synthesized sense and antisense as well as mismatched K14 oligonucleotide gene fragments were introduced into KCs by means of liposome. Blank control group were set. The effects of antisense oligonucleotide on the cell cycle,K14 gene and protein expressions of KCs were detected by flow cytometer, reverse transcription polymerase chain reaction and SABC methods.MAIN OUTCOME MEASURES: Effect of oligonucleotide transfecting human KCs on the proliferation of KCs and K14 expression.RESULTS: [1]The electrophoresis of reverse transcription polymerase chain reaction products: Specific K14 gene band appeared in each group, and K14 gene expression in the antisense group was significantly lower in the sense group,missense group and blank control group. K14/β-actin value was similar among sense group, missense group and blank control group (P ＞ 0.05), But K14/β-actin value was significantly lower in the antisense group than in the above-mentioned 3 groups (F =47.554, P ＜ 0.01). ②K14 protein expression detected by immunohistochemical method:K14 was expressed in all the cultured KCs at different levels, and was obviously reduced after antisense oligonucleotide being added. 20 μmol/L antisense oligonucleotide could markedly inhibit K14 expression; K14 expression did not change in the control group. ③ DNA level change detected by flow cytometer: After being treated by K14 antisense oligonucleotide for 48 hours, human epidermal KCs were significantly increased at G1 stage (74.6%), and were markedly decreased at S stage (19.4%). Such changes were not found in the antisense group, missense group and blank control group.CONCLUSION: Antisense oligonucleotide can specifically inhibit K14 synthesis, and thereby, inhibit the proliferation of human KCs.