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Objective:To investigate the effect of small extracellular vesicles (sEVs) derived from mesenchymal stem cells (MSCs) in mouse model of retinal light injury and the possible mechanism.Methods:Human umbilical cord derived MSCs were identified by flow cytometry.Supernatants of passage 3-5 MSCs were collected.sEVs were harvested by ultracentrifugation and were identified by transmission electron microscopy.Sixty-five healthy female SPF-grade BALB/c mice aged 8-10 weeks were randomly divided into normal group (17 mice), phosphate buffered saline (PBS) group (24 mice) and sEVs group (24 mice). Mice in PBS and sEVs groups were intravitreally injected with 2 μl of PBS and sEVs, respectively, and were exposed to 930 lx blue light for 6 hours.No intervention was administered to the normal group.Three days after lighting, mice retinal structure was observed by hematoxylin-eosin staining.Apoptotic retinal cells were detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL). Retinal function was tested by electroretinogram.Differentially expressed mRNAs between PBS group and sEVs group were assayed by mRNA transcriptome sequencing and were analyzed through KEGG cluster analysis.The differential mRNAs were verified via real-time quantitative PCR.The study protocol was approved by the Animal Ethics Committee of Tianjin Medical University Eye Hospital (No.TJYY20201221035).Results:MSCs were positive for CD90 and CD105, negative for CD34 and CD45.The extracted MSC-sEVs showed a bilayer membrane vesicle with a diameter of 80-140 nm.Hematoxylin-eosin staining showed the arrangement of photoreceptor nuclei was disordered in outer nuclear layer in PBS group.The disorder of photoreceptor nuclei arrangement of sEVs group was slighter than that of PBS group.The apoptotic cell number of sEVs group was (14.60±4.04)/visual field, which was lower than (24.00±8.52)/visual field of PBS group, with a statistically significant difference ( t=2.37, P<0.05). The a-wave amplitude of sEVs group was (64.38±16.70)μV, which was higher than (16.78±6.37) μV of PBS group, showing a statistically significant difference ( P<0.05). The b-wave amplitudes of PBS and sEVs groups were (132.40±39.41) μV and (154.86±34.08) μV, respectively, which were lower than (338.38±27.41) μV of normal group, and the differences were statistically significant (both at P<0.05). A total of 110 differentially expressed mRNAs were detected.There were 109 downregulated mRNAs in sEVs group.Differentially expressed mRNAs were mainly inflammation- and immune-related pathways.PCR showed that the expression level of C-C motif chemokine ligand 2, C-C motif chemokine receptor 2, leukotriene B4, leukocyte Ig-like receptor A6 and interleukin-1β in sEVs group were significantly decreased in comparison with PBS group (all at P<0.05). Conclusions:MSC-sEVs can ameliorate blue light-induced retinal structural and functional damage.The protective effect may be achieved through inhibiting inflammatory response.
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Objective To investigate the protective effect of butylphenyphthalein (NBP) on RPE apoptosis induced by H2O2.Methods The human RPE cell line (human ARPE-19 cell line) were used as the experimental cells and were divided as control group, model group, NBP group. Complete medium was used in control group. The model group was stimulated with 200 μmol/L H2O2 for 2 h, and the cells were cultured in complete medium. The NBP group was cultured with 200 μmol/L H2O2 and 1 μmol/L NBP for 2 h. After changing the medium, complete medium was combined with 1 μmol/L NBP to continue the culture of the cells. Cell viability were detected by MTT assay while the morphology of RPE were observed by HE staining. Moreover, Hoechst 33258 was used to detect RPE cell apoptosis. Mitochondrial membrane potential (JC-1) staining were performed to monitor changes in cell membrane potential and the characteristic change of apoptosis in RPE cells. Furthermore, 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) staining were used to analyze the effect of NBP treatment on the expression of ROS. The effect of NBP on the expression of Heme oxygenase-1(HO-1) was analyzed by cellular immunofluorescence and western blotting.Results The results of MTT assay showed that the cells were cultured for 24 and 48 hours, cell viability of control group (t=17.710, 13.760;P<0.0001,<0.0001) and treatment group (t=4.857, 9.225;P=0.0007,<0.0001) were stronger than that of model group, and the difference was statistically significant. HE staining and Hoechst33258 staining showed that compared with the control group, the number of cells in the model group was significantly less, and the cell morphology was incomplete. Compared with the model group, the number of cells in the treatment group was significantly increased, and the cell morphology was better. The results of JC-1 assay showed that the number of apoptotic cells in the model group was significantly higher than that in the control group, and the number of apoptotic cells in the treatment group was significantly lower than that in the model group. DCFH-DA staining showed that the ROS accumulation in the model group was more than that in the control group, and the ROS accumulation in the treatment group was less than that in the model group. Immunostaining observation showed that the HO-1 fluorescence intensity of the cells in the treatment group was significantly higher than that of the control group, and the difference was statistically significant (t=10.270,P=0.0005). Western blot analysis showed that NBP up-regulated the expression level of HO-1 in a time-dependent manner. The relative expression of HO-1 at 4, 8, and 12 h of NBP showed a clear increase trend compared with 0 h, and the difference was statistically significant (F=164.91,P<0.05).Conclusions Oxidative stress injury can down-regulate the viability of RPE cells and induce apoptosis. NBP can increase the antioxidant capacity of RPE cells, reduce cell damage and inhibit cell apoptosis by up-regulating HO-1 expression.
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Objective To observe the protective effect of dl-3-n-Butylphthalide (NBP) on apoptosis of retinal Müller cells induced by hydrogen peroxide (H2O2).Methods Human retinal Müller cells cultured in vitro were divided into normal control group,model group (H2O2 group) and experimental group (H2O2+NBP group).The cells in the H2O2 group and H2O2+NBP group were cultured with 200 μ mol/L H2O2 for 2 h.Then the culture solution of the H2O2 group replace with complete medium and the H2O2+NBP group replace with complete medium containing 1 tmol/L NBP.The normal control group was a conventional cultured cells.Müller cells were identified by immunofluorescence staining.Hematoxylin-eosin (HE) staining was used to observe the apoptosis morphological changes.MTT assay was used to detect the activity of of retinal Müller cells after after 24 h and 48 h of NBP intervention.Hoechst33258 staining was used to observe the apoptosis.LIVE/DEAD (R)cell activity/cytotoxicity kit was used to detect cell viability.Dichlorofluorescein diacetate (DCFH-DA) + endoplasmic reticulum (ER) red fluorescent probe (ER-Tracker Red) double staining was used to observe the expression level of reactive oxygen species (ROS) in ER of cells.One-way ANOVA combined with Dunnett statistical method were used for data analysis.Results HE staining showed that the number of cells in H2O2+NBP group was higher than that in H2O2 group.MTT assay showed that after 24 h and 48 h of NBP intervention,the differences in cell viability between the normal control group and the H2O2 group,the H2O2 group and the H2O2+NBP group were statistically significant (t=28.96,3.658,47.58,20.33;P＜0.001,0.022).The results of Hoechst33258 showed that the nuclear nucleus of a few cells in the H2O2+NBP group was crescent-shaped and the nuclear fragmentation was reduced,and the blue fluorescence of the remaining cells was uniform.The LIVE/DEAD ~ cell activity/cytotoxicity kit showed that the number of dead cells with red fluorescence in the H2O2 group increased significantly,and the number of viable cells with green fluorescence decreased significantly.In the H2O2+NBP group,the number of viable cells with green fluorescence increased,and the number of dead cells with red fluorescence decreased.The double staining results of DCFH-DA+ER-Tracker Red showed that the green fluorescence intensity of H2O2 group was significantly enhanced;the green fluorescence intensity of H2O2+NBP group was lower than that of H2O2 group.Conclusion NBP alleviates H2O2-induced apoptosis of human retinal Müller cells by inhibiting ROS production.
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Objective To analyze of the effect of artificial lengthening femoral head replacement in elderly patients with stage Ⅰ of unstable femoral intertrochanteric fracture.Methods 203 patients with stage Ⅰ of unstable femoral intertrochanteric fracture were selected as the research object,and they were taken artificial lengthening femoral head replacement,among which 65caese were male,female in 138 cases.The Harris scoring,SF-36,VAS pain scores on admission,2 weeks after operation,postoperative follow-up limb were counted,and the pain of the affected limb and the hip scores were compared amond 3 time periods.Results All 203 cases of senile patients with follow-up,average operation time was 83.64 minutes,the intraoperative blood loss was 355.41mL.The curative effect was evaluated according to the Harris score,SF-36 and VAS pain scoring criteria,and the Harris scores of the affected limbs at admission,at 2 weeks after the operation and after the follow-up were (28.26 ± 5.50) points,(68.26 ±5.50) points,(93.13 ± 5.31) points,respectively,the differences were statistically significant (t =-71.27,-1 397.55,-46.07,all P ＜ 0.01);The VAS pain scores were (8.19 ± 0.48) points,(3.53 ± 0.71) points,(0.23 ± 0.42) points,respectively,the differences were statistically significant (t =88.06,324.17,60.84,all P ＜ 0.01).The sf-36 scores:physiological [(8.35 ± 1.24) points,(15.23 ± 2.17) points,(19.21 ± 2.12) points],social/family [(7.01 ±1.13) points,(14.12 ± 2.12) points,(19.85 ± 2.24) points],emotional [(4.83 ± 1.01) points,(10.12 ±1.22)points,(14.87 ± 1.32) points],function [(6.35 ± 1.21) points,(13.67 ± 1.87) points,(16.81 ±2.12) points],additional focus [(8.85 ± 1.45) points,(16.38 ± 2.12) points,(20.21 ± 2.42) points],total quality of life [(47.35 ± 4.76) points,(74.69 ± 5.87) points,(89.21 ± 6.12) points],the differences were statistically significant(-39.77,-62.92,-20.21,-44.87,-71.89,-26.79,-45.04,-89.01,-38.25,-45.79,-63.41,-15.29,-45.20,-60.39,-17.54,-52.12,-76.49,-22.58,all P＜0.O1).Conclusion Artificial lengthening femoral head replacement in elderly patients with stage Ⅰ of unstable femoral intertrochanteric fracture has good clinical effect,intraoperative high safety,less postoperative complications,postoperative limb functional recovery is good,and it is worthy of clinical promotion and application.
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Objective To investigate the effect of exogenous atrial natriuretic peptide on airway inflammation in allergic asthma mice model and its mechanism.Methods A total of 48 SPF 48 BALBc mice were selected and randomly divided into the normal group (equal quantity normal saline intervention),model group (equal quantity normal saline intervention),experimental group A (exogenous atrial natriuretic peptide 0.5 μg/g) and experimental group B(exogenous atrial natriuretic peptide 0.5 μg/g-+-A71915).The model group,experimental group A and B were given the corresponding treatment measures after successfully constructing the model,at 24 after the last provocation test,serum and bronchoalveolar lavage fluid in each group were collected for detecting inflammation factors.The HE staining was used to observe the pathological changes of lung tissue,the expression level of GATA3 protein in lung tissue was detected by Western blot technology.Results The blood EOS,lymphocyte and neutrophil ratio in the model group were higher than those in the normal group (P＜0.05),which in the experimental group A were higher than those in the model group and experimental group B (P＜0.05);bronchoalveolar lavage fluid EOS,lymphocytes,neutrophil ratio,IL-6,TNF-α levels in the model group were higher than those in the normal group (P＜0.05),alveolar lavage fluid EOS,lymphocyte,neutrophil count,IL-6 and TNF-α levels in the experimental group A were higher than those in the model group and experimental group B (P＜0.05);the expression level of GATA3 protein in lung tissue of the model group was higher than that in the normal group(P＜0.05),the expression level of lung tissue GATA3 protein in the experimental group A was higher than that in the model group and experimental group B (P＜0.05).Conclusion Exogenous atrial natriuretic peptide can further aggravate airway inflammation reaction in allergic asthma mice model,which is related to the activation of inflammatory cells,the release of inflammatory cytokines and the increase of GATA3 protein expression in lung tissue.
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Objective To explore the best time interval to extubate catheter for the postoperative gastric cancer patients,and provide the basis for early extubation after operation.Methods To collect 108 cases with gastric cancer in Department of Forensic Surgery of Yuncheng Central Hospital from March 2013 to February 2014 with indwelling catheter preoperatively.According to the admission sequence,they were divided into the observation group and the control group with 54 cases in each group,the observation group began clipping catheter in 10-15 hours after operation,extubating catheter within 24 hours after operation;the control group began clipping catheter on the second day after operation,and extubating catheter during 48-72 hours after operation.The time of extubation,urinary tract irritation symptoms,resetting catheters and the incidence of early postoperative ambulation in patients of two groups were observed.Results The incidence of urinary tract irritation was lower in the observation group than that of the control group [3.7％ (2/54) vs 16.7％(9/54)],the difference was statistically significant (x2=4.960,P＜0.05).Early ambulation compliance of the observation group was higher than that of the control group (complete compliance 52 vs.40,partial compliance 2 vs.8,no compliance 0 vs.6),the difference was statistically significant (x2 =10.565,P ＜0.05).Conclusions The method of extubation catheter within 24 hours after gastric canter operation can reduce the incidence of urinary tract irritation and improve early ambulation compliance.
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Objective To investigate the expression level and clinical significance of serum IL-18 and MCP-1 in patients with Chronic Obstructive Pulmonary Disease(COPD).Methods Serum IL-18 and MCP-1 levels were measured in healthy people( control group,30 cases) and in patients with COPD (acute stage group and stable stage group,30 cases in each group).At the same time,the level of serum CRP and forced expiratory volume in one second ( FEV1 ),the forced expiratory volume in one second/forced vital capacity ( FEV1/FVC％ ) were tested in the three groups.The variation.of the level of serum IL-18 and MCP-1 and the correlation between these index with lung function and CRP were analyzed.Results The levels of serum IL-18 and MCP-1 in acute stage group ( IL-18 [ 69.29 ± 16.14 ] ng/L,MCP-1 [ 123.95 ± 32.06 ] ng/L) were significantly higher than those in stable stage group ( IL-18 [ 53.15 ± 14.74 ] ng/L,MCP-1 [ 96.46 ± 18.24 ] ng/L) ( F =10.975,P ＜0.01 ),and also significantly higher than those in control group ( IL-18 [ 37.53 ± 14.55 ] ng/L,MCP-1 [60.86 ± 17.26] ng/L) ( F =18.101,P ＜ 0.01 ).In acute stage group,positive correlations were obtained among the level of serum IL-18,MCP-1 and CRP ( r =0.688,P ＜ 0.05 ; r =0.663,P ＜ 0.05 ; r =0.825,P ＜0.01 ),and they were correlated negatively with FEV1 and FEV1/FVC％ ( r =- 0.732,P ＜ 0.01 ;r =- 0.586,P ＜0.05 ).In stable stage group,the level of serum MCP-1 was correlated positively with the level of IL-18( r =0.674,P ＜ 0.05 ) and with the level of CRP ( r =0.719,P ＜ 0.05 ),but it was correlated negatively with FEV1 and FEV1/FVC％ ( r =-0.512,P ＜ 0.05 ;r =-0.557,P ＜ 0.05 ).Conclusion The serum IL-18 and MCP-1may be one of the inflammation markers in patients with COPD and may be associated with airway inflammation and limited airflow.