1.Construction of a micro-vascular network model in vitro by microfluidic technology for hemodynamic investigations
Yuan LI ; Beizhong LIU ; Fang GONG ; Chao YU ; Liyi HU
Military Medical Sciences 2014;(11):885-889
Objective The micro-vascular element plays a key role in the delivery of nutrients and the regulation of hemodynamic behavior, however, research is often hindered by ethical , economic and technological issues .Therefore, construction of a micro-vascular network in vitro will help to study the related pathological and physiological behavior in microvessels.Methods In this study, a micro-vascular element model with features of a micro-vascular network in vivo was designed based on the network structure of retinal arterioles .A micro-vascular network model in vitro, characterized by network asymmetry and the presence of both bifurcation-and side-branches , was developed by soft lithography technology . The developed microdevice allowed for the quantification of the cell -depletion layer ( CDL) thickness and hematocrit ( Ht) distribution within the microchannel networks .Results and Conclusion The study showed the potential of the developed in vitro model in revealing key hemodynamic features which have been detected for microvascular elements in vivo, including the relationships between CDL thickness , Ht and red blood cell distribution .The present study provides a new strategy and a technology for studying hemodynamics and microvascular system diseases in vitro.
2.Kainic acid-induced endoplasmic reticulum stress model
Lei YUAN ; Haixia ZHANG ; Shilei QIAN ; Bin XU ; Jiqin GONG ; Xianghua LIU ; Yuan TANG ; Huaxu YU
Chinese Journal of Tissue Engineering Research 2014;(36):5861-5867
BACKGROUND:Previous studies have shown that kainic acid injected into hippocampus can significantly upregulate the expression of excitatory KA1 subunit of the kainate receptor in the hippocampus, and endoplasmic reticulum stress markers, phosphorylation of the alpha subunit of eukaryotic initiation factor 2, accompanied by celldeath. OBJECTIVE:To explore the mechanism of endoplasmic reticulum stress after kainic acid is injected into the hippocampus.METHODS:0.15 nmol kainic acid was injected into the hippocampal CA1 region of 32 adult male Kunming mice, the injection time was 60 seconds. At different time points (1, 2, 3, 4, 5, 6, 8 and 12 hours) after kainic acid was injected, the Bederson score analysis was performed, and then the brain was harvested after cerebral perfusion. FJB staining of brain sections and immunofluorescence double labeled observation were also performed. RESULTS AND CONCLUSION:(1) At 3, 4, 5, 6, 8 hours after kainic acid injection, Bederson score showed severe injury of central nervous system function, and FJB staining showed the increased of celldeath in the hippocampus (P<0.05);At 1, 2, 12 hours after injection, Bederson score showed no obvious injury of central nervous system function, and FJB staining showed unobvious celldeath in the hippocampus (P>0.05). (2) According to the results of FJB staining, the brain sections were selected at 3, 8 hours for immunohistochemistry. The expressionlevels of KA1 receptors and endoplasmic reticulum stress marker P-eIF2αwere up-regulated at the same time after kainic acid was injected into hippocampus. Two single-staining KA1 and P-eIF2αimmunofluorescence images were synthesized into one over-lapped double-stained image, and two images overlapped, indicating that the up-regulated expression of KA1 and endoplasmic reticulum stress occurred in the same nerve cells. Kainic acid first up-regulated the excitatory receptor KA1 expression, which may cause cellendoplasmic reticulum dysfunction and result in the endoplasmic reticulum stress response, further promoting neuronal celldeath.
3.Effect of non-lethal H2O2-induced persistent oxidative injury on retinal pigment epithelial barrier
Hong-mei, ZHANG ; Yuan-yuan, GONG ; Xing-wei, WU ; Yu-hua, SHI
Chinese Journal of Experimental Ophthalmology 2012;30(3):234-238
BackgroundReactive oxygen intermediate products lead to the oxidative injury of cells.Retinal pigment epithelial(RPE) cells produce lots of reactive oxygen intermediate products during the swallow of out disc,but how this procedure cause the persistent oxidative injury of RPE cells is poorly understood.Objective The present study was to evaluate the effect of non-lethal H2 O2 -induced persistent oxidative injury on RPE barrier in vitro.MethodsARPE-19 cell links were inoculated on 96 well plate at the density of 8×104 cells/L and the cell climbing slice of 24 well at the density of 4× 104 cells/L.The cells were cultured in DMEM/F12 medium,and the cells cultured for 24 hours in free-serum medium were used in the experiment.0-0.6 mmol/L of H2O2 were added into the medium.Cellular viability was assessed using 3- ( 4,5-dimethylthiazol-2-yl ) -5- ( 3-carboxymethoxyphenyl ) -2- ( 4-sulfophenyl ) 2H-tetrazolium(MTS) assays.Transepithelial electrical resistance (TER) was used to detect cell monolayer forming time after cultureinTrsnswellchamber.Thepermeabilityof cellmonolayer was examinedbyrhodamine isothiocyanate-dextran transepithelial flux,and immunofluorescence was used to investigate the distribution of the junction protein zonula occludens (ZO-1).ResultsThe total difference was found in the cell vitality(A490) among the different concentrations of H2 O2 ( F =991.501,P =0.000 ).Compared with 0 mmoL/L H2 O2 group,the A490 values was gradually lowed from 0.20 mmol/L H2O2 group to 0.60 mmol/L H2O2 group (P < 0.05 ).H2O2 at the concentrations of >0.20 mmol/L lowed the viability of RPE cells.The TER value was ( 24.9 ± 1.3 ) Ω · cm2 in 11 days,( 17.8± 1.4)Ω · cm2 in 7 days after inoculation on transwell chamber,showing a significant difference between them (t=5.228,P=0.014).RPE formed the stable tight junction on day 15 with the TER value (25.9±0.9 ) Ω · cm2.The leakage amount ( relative fluorescence intensity ) of the dextran was 255.39 ± 16.44 in non-H2 O2 control group,exhibiting a significant lowing in comparison with free-cell blank group (433.08±51.53)( t =12.515,P =0.006 ),and that of H2 O2 group was significant increased in comparison with non-H2 O2 control group ( t =14.412,P=0.005).Immunofluyorescence assay showed intact intercellular ZO-1 junction in non-H2O2 control group,but the breakage of ZO-1 junction was seen in H2O2 group.ConclusionsThe results indicate that non-lethal H2O2 can destroy RPE barrier and further lead to the persistent oxidative injury of RPE cells.
4.Changes in serum levels of antinuclear antibody, anti-double-stranded DNA antibody and anti-extractable nuclear antigens antibody before and after anti-tumor necrosis factor-α therapy in psoriatic patients
Suyun JI ; Yongfeng CHEN ; Xiao GONG ; Mei GU ; Yu WANG ; Liyan YUAN ; Bin YANG
Chinese Journal of Dermatology 2017;50(1):53-56
Objective To investigate changes in serum levels of antinuclear antibody(ANA), anti?double?stranded DNA(dsDNA)antibody and anti?extractable nuclear antigen(ENA)antibody before and after anti?tumor necrosis factor?α(TNF?α)therapy in psoriatic patients. Methods Clinical data obtained from 32 patients with psoriasis were analyzed retrospectively. Of the 32 patients, 13 received intravenous injection of 5 mg/Kg infliximab at week 0, 2, 6 for 3 sessions, then once every 8 weeks(infliximab group), while other 19 received subcutaneous injection of 25 mg etanercept twice every week(etanercept group). The treatments in the 2 groups both lasted more than 3 months. Serum levels of ANA, anti?dsDNA antibody and anti?ENA antibody and changes of clinical symptoms were detected and observed respectively before each treatment in the infliximab group, as well as every 3- 6 months in the etanercept group. The 75%reduction in psoriasis area and severity index(PASI75)and disease activity score of 28 joints(DAS28) were used to evaluate clinical efficacy. Serum levels of ANA, anti?dsDNA antibody and anti?ENA antibody were measured by indirect immunofluorescence(IIF)assay, Western blot analysis combined with enzyme?linked immunosorbent assay(ELISA), and Western blot analysis, respectively. Results After 3?month treatment, the 32 patients achieved clinical remission to different extents. Of 32 patients receiving anti?TNF?αtherapy, 7(21.9%)developed new autoantibodies. Concretely speaking, 4 patients in the infliximab group developed autoantibodies in 8.3 ± 5.1 months, including 3 cases positive for ANA and 3 for anti?ENA antibody. Three patients in the etanercept group developed autoantibodies in 9.0 ± 3.0 months, including 3 cases positive for ANA and 1 for anti?ENA antibody. Conclusion Partial patients with psoriasis may develop autoantibodies after anti?TNF?αtherapy.
6.Effect of electro-acupuncture on Nrf2 expression in renal tissues of rabbits with endotoxic shock-induced acute kidney injury: relationship with p38MAPK signaling pathway
Man WANG ; Lirong GONG ; Jianbo YU ; Xinshun CAO ; Yuan ZHANG ; Lili WU ; Jia SHI
Chinese Journal of Anesthesiology 2014;34(8):1012-1016
Objective To investigate the effect of electro-acupuncture (EA) on nuclear factor E2-related factor 2 (Nrf2) expression in the renal tissues of rabbits with endotoxic shock-induced acute kidney injury (AKI) and the relationship with p38 mitogen-activated protein kinase (p38MAPK) signaling pathway.Methods Seventy male New Zealand white rabbits,weighing 1.5-2.0 kg,aged 2 months,were randomized into 7 groups (n =10 each) using a random number table:normal control group (C group),endotoxic shock-induced AKI group (AKI group),EA + endotoxic shock-induced AKI group (EA group),non-acupoints + endotoxic shock-induced AKI group (SA group),EA + endotoxic shock-induced AKI + specific p38MAPK blocker SB203580 group (EAS group),SB203580 group (S group),and ethanol group (A group).EA (intensity 1-2 mA,frequency 2/100 Hz,wave length 0.2-0.6 ms) of Zusanli and Shenyu lasting for 15 min was performed once a day for 5 consecutive days in EA and EAS groups.In SA group,EA was performed at the points 0.5 cm lateral to the acupoints of bilateral Zusanli and Shenyu using the parameters of EA mentioned above.At 24 h after the last EA,endotoxic shock-induced AKI was induced by injection of lipopolysaccharide (LPS) 5 mg/kg (in 2 ml normal saline) in AKI,EA,SA and EAS groups,while the equal volume of normal saline was given in the other groups.At 30 min before the model was established,5/μmol/kg SB203580 (in 0.5 ml ethanol) was injected intravenously in EAS and S groups,while ethanol 0.5 ml was given in A group and the equal volume of normal saline was given in the other groups.Blood samples were obtained at 6 h after administration of LPS or normal saline for determination of serum urea nitrogen (BUN) and creatinine (Cr) concentrations.The animals were sacrificed and kidney specimens were obtained for microscopic examination of pathological changes which were scored and for measurement of Nrf2 protein expression and phosphorylation of p38MAPK (by Western blot) and Nrf2 mRNA expression (using fluorescent quantitative PCR).Results Compared with C group,the pathological score and serum BUN and Cr concentrations were significantly increased,and Nrf2 mRNA and protein expression was up-regulated in AKI,EA,SA and EAS groups,the phosphorylation of p38MAPK was increased in AKI,EA and SA groups,and no significant changes were found in the parameters mentioned above in S and A groups.Compared with AKI group,the pathological score and serum BUN and Cr concentrations were significantly decreased,and Nrf2 mRNA and protein expression was up-regulated in EA and EAS groups,the phosphorylation of p38MAPK was increased in EA group,the phosphorylation of p38MAPK was decreased in EAS group,and no significant changes were found in the parameters mentioned above in SA group.Compared with EA group,the pathological score and serum BUN and Cr concentrations were significantly increased,Nrf2 mRNA and protein expression was down-regulated,and the phosphorylation of p38MAPK was decreased in EAS group.Conclusion The mechanism by which EA mitigates endotoxic shock-induced AKI may be related to activation of p38MAPK signaling pathway and up-regulation of Nrf2 expression in renal tissues of rabbits.
7.Role of large conductance calcium-activated potassium channels in vascular hyporesponsiveness in rats with obstructive jaundice
Yawei YUAN ; Yue LONG ; Long WANG ; Wuxiang GONG ; Jinqiang HUANG ; Weifeng YU
Chinese Journal of Anesthesiology 2015;35(6):732-735
Objective To evaluate the role of large conductance calcium-activated potassium (BKCa) channels in vascular hyporesponsiveness in rats with obstructive jaundice.Methods Eighteen male Sprague-Dawley rats,weighing 180-200 g,were randomly divided into 3 groups (n =6 each) using a random number table:control group (group C),sham operation group (group S),and bile duct ligation group (group BDL).Obstructive jaundice was produced by common bile duct ligation.At 7 days after surgery,blood samples were collected for determination of the levels of serum total bilirubin (TBL),direct bilirubin (DBL),indirect bilirubin (IBL),alanine aminotransferase (ALT),and aspartate aminotransferase (AST).Thoracic aortic rings were prepared,and the endothelium was removed.The aortic rings were sequentially perfused with different concentrations of norepinephrine (NE) and sodium nitroprusside (SNP),and the maximum amplitude of contraction and dilatation of aortic rings was recorded.The aortic rings were then perfused with BKCa channel blocker Chtx with the final concentration of 10 7 mol/L,followed by perfusion with different concentrations of NE and SNP again,and the maximum amplitude of contraction and dilatation of aortic rings was recorded under each concentration.The percentage of maximum contraction and dilatation (maximum amplitude after Chtx administration÷maximum amplitude before Chtx administration× 100%) was calculated.Results Compared with C and S groups,the levels of TBL,DBL,IBL,ALT and AST in serum were significantly increased,the maximum amplitude of NE-induced contraction of aortic rings was decreased,and the percentage of the maximum NE-induced dilatation of aortic rings was increased,the maximum amplitude of SNP-induced contraction of aortic rings was increased,and the percentage of the maximum SNP-induced dilatation of aortic rings was decreased in group BDL.Conclusion Excessivc opening of BKCa channels may be involved in the mechanism of vascular hyporesponsiveness in rats with obstructive jaundice.
8.The perioperative nursing of microsurgical denervation of the spermatic cord to treat idiopathic chronic orthialgia
Yanwen GONG ; Caimei ZHOU ; Shaoqiong LUO ; Yu LI ; Shanyan YUAN ; Meiqiong ZOU
Chinese Journal of Practical Nursing 2013;29(26):26-27
Objective To summarize the perioperative nursing experience of microsurgical denervation of the spermatic cord to treat idiopathic chronic orthialgia.Methods We retrospectively analyzed the clinical record data of two patients with idiopathic chronic orthialgia,who received microsurgical denervation of the spermatic cord in our hospital,including pre-and postoperative symptom scores,evaluation of mental state and wound nursing.Results Both patients got complete pain relief and were discharged one week after operation.No would infection and mental fluctuation was noted.Conclusions The reasonable individual perioperative nursing is an elemental component of recovery for patients with chronic orthialgia who experienced microsurgical management.
9.Effect of c-AMP-protein kinase A on up-regulation of heme oxygenase-1 expresion during lipopolysac-charide-induced acute lung injury in rats
Dongmei MA ; Lirong GONG ; Jianbo YU ; Yuan ZHANG ; Shuan DOGN ; Li LI ; Daquan LIU
Chinese Journal of Anesthesiology 2012;(10):1267-1270
Objective To evaluate the role of c-AMP-protein kinase A (cAMP-PKA) on the up-regulation of heme oxygenase-1 (HO-1) expression during lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats.Methods Forty-eight healthy male Sprague-Dawley rats,weighing 180-220 g,aged 2.5-3.0 months,were randomly divided into 4 groups (n =12 each)∶ normal control group (group C),ALI group (group ALI),H89 +ALI group (group H + ALI) and H89 group (group H).In group C,normal saline (solvent for LPS) 0.5 ml was injected via the femoral vein and normal saline (solvent for H89) 0.5 ml was injected subcutaneously 2 h later.In group ALI,10 mg/kg LPS 0.5 ml was injected via the femoral vein and normal saline 0.5 ml was injected subcutaneously 2 h later.In group H +ALI,10 mg/kg LPS 0.5 ml was injected via the femoral vein and 5 mg/kg H89 0.5ml was injected subcutaneously 2 h later.In group H,normal saline 0.5 ml was injected via the femoral vein and 5 mg/kg H89 0.5 ml was injected subcutaneously 2 h later.The rats were then sacrificed at 6 h after iv injection of LPS and the lungs were removed for microscopic examination and lung water content.The pathological changes of the lung were scored.The expression of HO-1 and PKA (by Western blot) and HO-1 mRNA (by RT-PCR) was detected.Results Compared with group C,the pathological score and lung water content were significantly increased,and the expression of HO-1,PKA and HO-1 mRNA was up-regulated in groups ALI and H +ALI (P <0.05),and no significant change was found in the parameters mentioned above in group H (P > 0.05).The pathological score and lung water content were significantly higher,and the expression of HO-1,AP-1 and HO-1 mRNA was significantly lower in group H + ALI than in group ALI (P < 0.05).Conclusion Activation of signaling pathway c-AMP-PKA is involved in the up-regulation of HO-1 expression during LPS-induced ALI in rats.
10.Effects of electro-acupuncture at Zusanli and Feishu on endotoxin shock-induced acute lung injury in rabbits
Shuan DONG ; Xiaoqing LUO ; Jianbo YU ; Lirong GONG ; Yuan ZHANG ; Man WANG ; Daquan LIU ; Xinshun CAO
Chinese Journal of Anesthesiology 2012;32(1):103-106
Objective To evaluate the effect of electro-acupuncture (EA) at Zusanli and Feishu on endotoxin shock-induced acute lung injury in rabbits.Methods Sixty healthy male New Zealand white rabbits aged 2 months weighing 1.5-2.0 kg were randomly divided into 6 groups (n =10 each):group sham operation (group S); group zinc protoporphyrin-Ⅸ (ZnPP-Ⅸ) (group Z); group lipopolysaccharide (LPS) (group L); group LPS + EA (group EL) ; group LPS + sham EA (group SEL) and group LPS + EA + ZnPP-Ⅸ (group ELZ).The animals were anesthetized with intraperitoneal 10% chloral hydrate 400 mg/kg and tracheostomized.The animals kept spontaneous breathing.Right internal carotid artery was cannulated for BP monitoring.Ear vein was cannulated for drug administration.LPS 5 mg/kg was injected iv in groups L,EL,SEL,ELZ.Endotoxin shock was confirmed by decrease in BP by 20 % of the baseline value and PaO2/FiO2 ≤ 300.ZnPP-Ⅸ (heme oxygenase (HO-1 ) inhibitor)10μmol/kg was injected intraperitoneal at 2 h after LPS injection in groups Z and ELZ.Bilateral 15 min EA stimulation of Zusanli and Feishu ( according to atlas of animal acu-points) was performed once a day for 5 days before LPS administration in groups EL and ELZ.The animals were sacrificed by blood-letting at 6 h after LPS administration.The lungs were removed for microscopic examination (0 =no injury,4 =most severe injury),detection of alveolar epithelial cell apoptosis (by TUNEL) and determination of HO-1 protein and mRNA expression.Results LPS significantly increased lung injury scores,alveolar epithelial cell apoptosis index (the number of apoptotic cells/total cells) and HO-1 protein and mRNA expression.EA significantly attenuated lung injury and alveolar epithelial cell apoptosis induced by LPS and further increased the expression of HO-1 protein and mRNA in group EL as compared with group L.The protective effects of EA was counteracted by ZnPP- Ⅸ in group ELZ.Conclusion EA at Zusanli and Feishu can attenuate endotoxin shock-induced lung injury by up-regulation of HO-1 expression and inhibiting alveolar epithelial cell apoptosis in the lung.