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Objective:To evaluate the role of has_circ_0008039 and miR-484 in oxygen-glucose deprivation/reoxygenation (OGD/R) injury in SK-N-SH cells and the relationship with Fis1.Methods:SK-N-SH cells were cultured in vitro to logarithmic growth stage and divided into 5 groups ( n=25 each) according to the random number table method: control group (group C), OGD/R group, has_circ_0008039 siRNA group (group S), hsa_circ_0008039 over-expression group (group E) and has_circ_0008039 siRNA plus miR-484 inhibitor group (group S+ I). Cells were cultured in normal condition in group C. In S, E and S+ I groups, after the cells were transfected with hsa_circ_0008039 siRNA, has_circ_0008039 over-expression vector, hsa_circ_0008039 siRNA and miR-484 inhibitor, the cells were subjected to oxygen-glucose deprivation for 12 h followed by 24 h restoration of O 2-glucose supply to develop the OGD/R model.At 24 h of restoration of O 2-glucose supply, the cell viability and amount of lactic dehydrogenase (LDH) released were measured using CCK-8 assay, the expression of hsa_circ_0008039, miR-484 and Fis1 mRNA was detected using real-time polymerase chain reaction, and the expression of Fis1 was detected by Western blot.A dual-fluorescein experimental report was used to verify the targeting relationship between hsa_circ_0008039 and miR-484. Results:Compared with group C, the cell viability was significantly decreased, and the amount of LDH released was increased in the other 4 groups, the expression of hsa_circ_0008039 and Fis1 was significantly up-regulated, and the expression of miR-484 was down-regulated in OGD/R and E groups, the expression of hsa_circ_0008039 and Fis1 was significantly down-regulated, and miR-484 was up-regulated in group S, and the expression of hsa_circ_0008039 and miR-484 was significantly down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.05). Compared with group OGD/R, the cell viability was significantly decreased, and the amount of LDH released was increased in E and S+ I groups, the cell viability was significantly increased, and the amount of LDH released was decreased in group S, the expression of hsa_circ_0008039 and Fis1 was significantly up-regulated, and the expression of miR-484 was down-regulated in group E, the expression of hsa_circ_0008039 and Fis1 was significantly down-regulated, and the expression of miR-484 was up-regulated in group S, and the expression of hsa_circ_0008039 and miR-484 was significantly down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.05). Compared with group S, the cell viability was significantly decreased, the amount of LDH released was increased, the expression of miR-484 was down-regulated, and the expression of Fis1 was up-regulated in group S+ I ( P<0.01). The dual-fluorescein experimental report verified that miR-484 was the target of hsa_circ_0008039 which binded to miR-484 specifically. Conclusions:has_circ_0008039 is involved in OGD/R injury in SK-N-SH cells by targetedly binding to miR-484, which is associated with up-regulation of Fis1 expression.
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Objective:To evaluate the role of miR-20a-5p in M1 microglia aggravating oxygen-glucose deprivation and restoration (OGD/R)-induced injury to neurons and the relationship with mitofusin2 (MFN2).Methods:The well-growing BV2 microglia (M0 type) were polarized into M1 phenotype by lipopolysaccharide (100 ng/ml) and IFN-γ (20 ng/ml) and identified by quantitative real-time polymerase chain reaction and immunofluorescence.The well-growing N2a cells were divided into 6 groups ( n=6 each) by the random number table method: control group (group C), OGD/R group, M0 microglia co-culture group (group M0), M1 microglia co-culture group (group M1), miR-20a-5p inhibitor transfection group (group I) and negative control group (group NC). The cells were routinely cultured in group C, and the cells were subjected to OGD for 3 h followed by restoration of oxygen-glucose supply to develop the model of OGD/R injury in group OGD/R.The cells were subjected to OGD for 3 h and were co-cultured with M0 microglia for 24 h during restoration of oxygen-glucose supply in group M0.The cells were subjected to OGD for 3 h and were co-cultured with M1 microglia for 24 h during restoration of oxygen-glucose supply in group M1.In group I and group NC, cells were transfected with miR-20a-5p inhibitor and negative control miRNA into M1 microglia, respectively, and N2a cells were subjected to OGD for 3 h and co-cultured with M1 microglia for 24 h during restoration of oxygen-glucose supply.The cell viability was determined by cell counting kit-8 assay, amount of lactate dehydrogenase (LDH) released was determined, the expression of miR-20a-5p and MFN2 mRNA was detected by quantitative real-time polymerase chain reaction, and MFN2 expression was detected by Western blot. Results:Compared with group C, the cell viability was significantly decreased, the amount of LDH released was increased, and the expression of MFN2 protein and mRNA was down-regulated in the other five groups, miR-20a-5p expression was significantly up-regulated in OGD/R, M0 and M1 groups, and miR-20a-5p expression was significantly down-regulated in group I ( P<0.05). There were no significant differences in the cell viability, amount of LDH released, and expression of miR-20a-5p, MFN2 protein and mRNA between group OGD/R and group M0 ( P>0.05). Compared with group OGD/R and group M0, the cell viability was significantly decreased, the amount of LDH released was increased, and the expression of MFN2 protein and mRNA was down-regulated, and miR-20a-5p expression was up-regulated in group M1 ( P<0.05). Compared with group M1, the cell viability was significantly increased, the amount of LDH released was decreased, the expression of MFN2 protein and mRNA was up-regulated, and miR-20a-5p expression was down-regulated in group I ( P<0.05). Conclusions:The mechanism by which M1 microglia aggravates OGD/R-induced damage to N2a cells may be related to the up-regulation of miR-20a-5p expression in M1 microglia and the inhibition of MFN2 expression in N2a cells.
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Objective:To evaluate the relationship between microRNA-93-5p and mitochondrial fusion protein-2 (Mfn2) in mouse nerve cells subjected to oxygen-glucose deprivation and reoxygenation (OGD/R).Methods:Mouse neuroblastoma cells were cultured in vitro to logarithmic growth phase.Experiment Ⅰ Cells were divided into 5 groups ( n=20 each) by the random number table method: control group (group C), group OGD/R, miR-93-5p inhibitor group (group I), siRNA-mfn2 plus miR-93-5p group (group siMfn2+ I) and miR-93-5p negative control group (group NC). Oxygen-glucose deprivation: the cells were cultured for 3 h in a low-glucose balanced salt solution at 37 ℃ in an environment of 5% CO 2-95% N 2.Restoration of oxygen and glucose: the cells were cultured in normal medium at 37℃ in 5% CO 2-95% air for 24 h. Group I, group siMfn2+ I and group NC were transfected with miR-93-5p inhibitor, miR-93-5p inhibitor plus siRNA-mfn2 and negative control miRNA, respectively, at 48 h before the OGD/R model was developed.Cell viability was measured by CCK-8 assay.Cell apoptosis rate was measured by flow cytometry.Quantitative real-time polymerase chain reaction was used to detect the expression of miR-93-5p and Mfn2 mRNA.Western blot was used to detect Mfn2 protein expression.Experiment Ⅱ The wild-type (WT)-Mfn2 and mutant (MUT)-Mfn2 were constructed and transfected into neuroblastoma cells with miR-93-5p mimic and miR-93-5p blank control (miR-93-5pNC), respectively.The cells were divided into 4 groups ( n=5 each) after 48 h of transfection by the random number table method: miR-93-5p NC-WT-Mfn2 co-transfection group, miR-93-5p mimic-WT-Mfn2 co-transfection group, miR-93-5p NC-MUT-Mfn2 co-transfection group, and miR-93-5p mimic-MUT-Mfn2 co-transfection group.The activity of luciferases was measured by double luciferase assay. Results:Experiment Ⅰ Compared with group C, the cell viability was significantly decreased, the apoptosis rate of cells was increased, the expression of miR-93-5p was up-regulated, and the expression of Mfn2 protein and mRNA was down-regulated in the other gorups ( P<0.05). Compared with group OGD/R or group NC, the cell viability was significantly increased, the apoptosis rate of cells was decreased, miR-93-5p expression was down-regulated, and the expression of Mfn2 protein and mRNA was up-regulated in group I ( P<0.05). Compared with group I, the cell viability was significantly decreased, the apoptosis rate of cells was increased, and the expression of Mfn2 protein and mRNA was down-regulated in group siMfn2+ I ( P<0.05). Experiment Ⅱ Compared with miR-93-5p NC-WT-Mfn2 co-transfection group, the luciferase activity was significantly decreased in miR-93-5p mimic-WT-Mfn2 co-transfection group ( P<0.05). There was no significant difference in luciferase activity between miR-93-5p NC-MUT-Mfn2 co-transfection group and miR-93-5p mimic-MUT-Mfn2 co-transfection group ( P>0.05). Conclusions:The miR-93-5p expression is up-regulated, which further targetedly down-regulates the expression of Mfn2, and this may be a mechanism of OGD/R in mouse nerve cells.
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Objective:To evaluate the role of transient receptor potential melastatin 2 (TRPM2)-calcineurin A (CnA)-dynamin-related protein 1 (Drp1) pathway in propofol-induced reduction of renal injury induced by hepatic ischemia-reperfusion (I/R) in mice.Methods:Twenty-four SPF male C57BL6 mice, aged 8 weeks, weighing 20-23 g, were divided into 4 groups ( n=6 each) using a random number table method: sham operation group (group S), hepatic I/R group (group IR), propofol group (group P) and TRPM2 agonist (ADPR) combined with propofol group (AP group). Hepatic I/R injury was induced by occluding the portal vein and hepatic artery supplying the left and middle lobes of the liver for 60 min followed by reperfusion in anesthetized rats.In group P, 0.2 ml normal saline was injected intraperitoneally at 1 h before establishing the model and 1% propofol 30 mg/kg was injected intraperitoneally at 30 min before establishing the model.In group AP, ADPR 10 mg/kg (in 0.2 ml of normal saline) was injected intraperitoneally at 1 h before establishing the model, and 1% propofol 30 mg/kg was injected intraperitoneally at 30 min before establishing the model.The equal volume of normal saline was given intraperitoneally at 1 h and at 30 min before establishing the model in group S and group IR.Blood samples were taken from the eyeballs for determination of the levels of serum urea nitrogen (BUN), creatinine (Cr), aminotransferase (ALT) and aspartate aminotransferase (AST) at 6 h of reperfusion.The animals were then sacrificed and the kidney tissues were taken, the ultrastructure of myocardial mitochondria was observed using transmission electron microscopy, the average diameter of mitochondria was calculated, and the expression of TRPM2, CnA, phospho-Drp1 Ser637 (p-Drp1 Ser637) and cleaved caspase-3 was detected (by Western blot). Results:Compared with group S, the concentrations of serum BUN and Cr were significantly increased, the expression of TRPM2, CnA and cleaved caspase-3 in kidney tissues was up-regulated, the expression of p-Drp1 ser637 was down-regulated, and the average diameter of mitochondria was shortened in IR, P and AP groups ( P<0.05). Compared with group IR, the concentrations of serum BUN and Cr were significantly decreased, the expression of TRPM2, CnA and cleaved caspase-3 in kidney tissues was down-regulated, the expression of p-Drp1 Ser637 was up-regulated, the average diameter of mitochondria was prolonged ( P<0.05), mitochondrial injury was attenuated, and no significant change was found in the serum ALT and AST concentrations in group P, and no significant change was found in concentrations of BUN and Cr in serum in group AP ( P>0.05). Compared with group P, concentrations of BUN and Cr in serum was significantly increased, the expression of TRPM2, CnA and cleaved caspase-3 in kidney tissues was up-regulated, the expression of p-Drp1 Ser637 in kidney tissues was down-regulated, and the average diameter of mitochondria was shortened ( P<0.05), and mitochondrial injury was accentuated in group AP. Conclusion:The mechanism of propofol-induced reduction of renal injury induced by hepatic I/R is related to inhibiting the expression of TRPM2 in kidney tissues, decreasing the level of intracellular CnA and inhibiting dephosphorylation of Drp1 Ser637 in mice.
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Objective@#To explore the prognostic value of human mitochondrial transcription termination factor 3 (hMTERF3) and forkhead box protein 3 (Foxp3) in non-small cell lung cancer (NSCLC).@*Methods@#The clinical data of 88 patients with NSCLC who were admitted to the Third Medical Center of PLA General Hospital from March 2017 to March 2018 were retrospectively analyzed. All patients were diagnosed by pathological puncture. The patients were followed-up by telephone for 12 months, and according to the prognosis, the patients were divided into good prognosis group and poor prognosis group. The pathological tissues were taken from all patients, and the expressions of hMTERF3 and Foxp3 proteins were detected by immunohistochemistry. The expressions of hMTERF3 and Foxp3 in the good prognosis group and the poor prognosis group were compared. Logistic regression model was used to analyze the risk factors of poor prognosis in patients with NSCLC.@*Results@#Of 88 patients, 61 patients (69.3%) had good prognosis and 27 patients (30.7%) had poor prognosis. The positive expression rate of hMTERF3 in the good prognosis group was 57.4% (35/61), which was significantly lower than that in the poor prognosis group (81.5%, 22/27) (χ 2= 4.766, P= 0.029). The positive expression rate of Foxp3 in the good prognosis group was 55.7% (34/61), which was significantly lower than that in the poor prognosis group (85.2%, 23/27) (χ 2= 7.113, P= 0.008). The proportions of patients with medium and high differentiation or stage Ⅰ- Ⅱ in the good prognosis group were 82.0% (50/61) and 68.8% (42/61), respectively, which were significantly higher than those in the poor prognosis group [48.15% (13/27) and 25.93% (7/27)] (both P < 0.05). Logistic regression analysis showed that the poor differentiation, stage Ⅲ-Ⅳ, hMTERF3-positive and Foxp3-positive were the risk factors for poor prognosis in NSCLC patients (all P < 0.05).@*Conclusions@#The positive expression rates of hMTERF3 and Foxp3 in patients with good prognosis are lower. The hMTERF3-positive and Foxp3-positive are risk factors for poor prognosis in NSCLC patients.
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Objective To investigate the relationship between glycoprotein synthase kinase-3 (GSK-3β) and mitochondrial cleavage protein (Drp-1) during diabetes mellitus-caused antagonization of cardioprotection induced by sevoflurane postconditioning in rats.Methods Clean-grade healthy male Sprague-Dawley rats,weighing 250-300 g,were used in this study.Diabetes mellitus was induced by high-fat and high-sucrose diet and intraperitoneal streptozotocin 30 g/kg.Forty rats with diabetes mellitus were divided into 5 groups (n =8 each) using a random number table method:ischemia-reperfusion (I/R) group,sevoflurane postconditioning group (SP group),sevoflurane postconditioning plus Drp1 inhibitor Mivi-1 group (SM group),sevoflurane postconditioning plus GSK-3β inhibitor SB216763 group (SB group) and sevoflurane postconditioning plus Mivi-1 plus SB216763 group (SMB group).Myocardial I/R was induced by 30 min occlusion of the left anterior descending branch of the coronary artery followed by 120 min reperfusion.The rats inhaled 2.5％ sevoflurane for 10 min starting from 5 min before reperfusion in SP,SM,SB and SBM groups.Mivi-1 1.2 mg/kg was injected via the caudal vein at 15 min before reperfusion in group SM.SB216763 0.2 mg/kg was injected via the caudal vein at 5 min before reperfusion in group SB.Mivi-1 1.2 mg/kg and SB216763 0.2 mg/kg were injected via the caudal vein at 15 and 5 min before reperfusion,respectively,in group SMB.Blood samples were collected from the abdominal aorta at 120 min of reperfusion for determination of serum cardiac troponin Ⅰ (cTnⅠ) concentrations.The rats were sacrificed and myocardial specimens were obtained from the apex for determination of the cell apoptosis (by TUNEL) and expression of caspase-3 (by Western blot),and apoptotic index (AI) was calculated.Results Compared with group I/R,no significant change was found in caspase-3 expression,AI or serum cTnⅠ concentrations (P＞0.05),and the pathological changes of myocardium were comparable in group SP,and the expression of caspase-3 was significantly down-regulated,and AI and serum cTnⅠ concentration were decreased (P＜0.05),and the pathological changes of myocardium were significantly attenuated in SM,SB and SMB groups.Compared with group SP,the expression of caspase-3 was significantly down-regulated,AI and serum cTnⅠ concentrations were decreased (P＜0.05),and the pathological changes of myocardium were significantly attenuated in SM,SB and SMB groups.Compared with group SMB or group SB,the expression of caspase-3 was significantly down-regulated,AI and serum cTnI concentrations were decreased (P＜0.05),and the pathological changes of myocardium were significantly attenuated in group SMB.Conclusion It is not a single regulatory relationship between GSK-3β and Drp-1 in the pathophysiological process of diabetes mellitus-caused antagonization of cardioprotection induced by sevoflurane postconditioning in rats.
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Objective To observe glomerular mesangial cells (GMCs) proliferation induced by IgA1 and the association with the expression of apoptosis-related proteins-B cell lymphoma-2 (Bcl-2),cysteine aspartic acid protease-3 (Caspase-3),cysteine aspartic acid protease-9 (Caspase-9) and with mitofusin 2 (Mfn2) in rat GMCs,to study the possible mechanism of valsartan inhibiting rat GMCs proliferation,and to provide a new direction for the mechanism of GMCs proliferation and intervention research in IgA nephrology (IgAN).Methods GMCs stimulated with IgA1 were cultured in vitro to detect cellproliferation with the cell counting kit-8 cell activity assay (CCK8).GMCs were divided into three groups:CG,TG and VG.The GMCs proliferation level was detected by the CCK8,using real-time PCR to detect Mfn2 expression and Western blotting to detect protein levels of Mfn2,Bcl-2,Caspase-3,and Caspase-9.Results Rat GMCs proliferated significantly after stimulation with IgA1,and IgA1 could obviously stimulate high expression of Bcl-2 in GMCs and down regulate the expression of Mfn2,Caspase-3,and Caspase-9.Valsartan could inhibit the proliferation of GMCs induced by IgA1 significantly,downregulate the expression of Bcl-2,and upregulate the expression of Mfn2,Caspase-3,and Caspase-9.Conclusions These results showed that the mechanism of action of valsartan in the treatment of lgAN is inhibiting the proliferation of GMCs.This mechanism may be associated with the regulation of apoptosis-related proteins,such as Mfn2,Bcl-2,Caspase-3,and Caspase-9.These findings may provide a new direction for the mechanism of GMCs proliferation and intervention research in IgAN.
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Objective To evaluate the role of 1-phosphatidylinositol 3-kinase/serine threonine kinase (PI3K/Akt) signaling pathway in carbon monoxide (CO)-induced up-regulation of mitochondrial fusion proteins in endotoxin-challenged alveolar epithelial cells of rats.Methods The rat alveolar epithelial cells were cultured in a 5％ CO2 cell culture incubator at 37 ℃ with F12K complete medium containing 10％ fetal bovine serum and 1％ green chain double antibody,and divided into 10 groups (n=5 each) according to the random number table method:control group (C group),endotoxin group (L group),lipopolysaccharide (LPS) plus exogenous CO-releasing agent CO-releasing-molecule-2 (CORM-2) group (L+CO group),LPS plus PI3K inhibitor LY294002 group (L+LY group),LPS plus CORM-2 plus LY294002 group (L+ CO+LY group),LPS plus inactive CO releasing agent iCORM-2 group (L+iCO group),LPS plus dimethyl sulfoxide (DMSO) group (L+D group),CORM-2 group (CO group),LY294002 group (LY group) and CORM-2 plus LY294002 group (CO+LY group).LPS 10 μg/ml was added to the culture medium in group L.CORM-2 100 μmol/L was added to the culture medium and 1 h later 10 μg/ml LPS was added in L+CO group.LY294002 25 μmol/L was added to the medium,and 1 h later LPS 10 μg/ml was added in L+LY group.In L+CO+LY group,25 μmol/L LY294002 was added to the culture medium,CORM-2 100 μmol/L was added 1 h later,and then LPS 10 μg/ml was added 1 h later.iCORM 100 μmol/L was added to the culture medium and 1 h later LPS 10 μg/ml was added in L+iCO group.In L+D group,the equal concentration of DMSO was added to the culture medium and 1 h later LPS 1 μg/ml was added.CORM-2 100 μmol/L was added to the culture medium in CO group.LY294002 25 μmol/L was added to culture medium in LY group.LY294002 25 μmol/L was added to the culture medium,and 1 h later CORM-2 100 μmol/L was added in CO+LY group.Cells were harvested after 24 h of incubation for measurement of the malondialdehyde (MDA) content,superoxide dismutase (SOD) activity and expression of heme oxygenase-1 (HO-1),phosphorylated Akt (p-Akt),mitochondrial fusion proteins mitofusion 1 (Mfn1),Mfn2 and optic atrophy 1 (OPA1) by Western blot.Results Compared with group C,the MDA content was significantly increased and SOD activity was decreased in L,L+CO,L+LY,L+CO+LY,L+iCO,L+D groups,and the expression of HO-1,Mfn1,Mfn2,OPA1 protein and p-Akt was significantly up-regulated in group L (P＜0.05).Compared with group L,MDA content was significantly decreased and SOD activity was increased in group L+CO,MDA content was significantly increased and SOD activity was decreased in group L+LY,the expression of HO-1,Mfn1,Mfn2,OPA1 protein and p-Akt was significantly up-regulated in group L+ CO,and the expression of HO-1,Mfn1,Mfn2,OPA1 protein and p-Akt was significantly down-regulated in group L+LY (P＜0.05).Compared with group L+CO,the MDA content was significantly increased,SOD activity was decreased,and the expression of HO-1,Mfn1,Mfn2,OPA1 and p-Akt was down-regulated in group L+CO+LY (P＜0.05).Conclusion The mechanism by which CO up-regulates the expression of mitochondrial fusion proteins in endotoxin-challenged alveolar epithelial cells is related to activating PI3K/Akt signaling pathway in rats.
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Objective To evaluate the role of protein kinase Cα (PKCα)/heme oxygenase-1 (HO-1) signaling pathway in endotoxin-induced damage to alveolar macrophages and the relationship with mitofusin-1 (Mfn1) in rats.Methods Rat alveolar macrophages NR8383 cells cultured in vitro were seeded in 96-well plates at a density of 1 × 104 cells/ml.NR8383 cells were divided into 5 groups (n =15 each)using a random number table:control group (group C),endotoxin challenge model group (group E),PKCα inhibitor Go6976 group (group G),PKCα agonist PMA group (group P) and dimethyl sulfoxide group (group D).NR8383 cells were stimulated with 10 μg/ml lipopolysaccharide (LPS) to establish the model of endotoxin challenge in alveolar macrophages.In G,P and D groups,cells were pretreated with 5 μmol/L Go6976,100 nmol/L PMA and 0.1％ dimethyl sulfoxide,respectively,for 30 min starting from 30 min before stimulation with LPS,and 10 μg/ml LPS was then given.The cells were collected after 24 h of incubation for measurement of malondialdehyde (MDA) and reactive oxygen species (ROS) contents,superoxide dismutase (SOD) activity and expression of PKCα,HO-1 and Mfn1 protein and mRNA (by fluorescent quantitative polymerase chain reaction or Western blot).Results Compared with group C,MDA and ROS contents were significantly increased,the SOD activity was decreased,the expression of PKCα and HO-1 protein and mRNA was up-regulated,and the expression of Mfn1 protein and mRNA was downregulated in E,G,P and D groups (P＜0.05).Compared with group E,MDA and ROS contents were significantly increased,the SOD activity was decreased,and the expression of PKCα,HO-1 and Mfn1 protein and mRNA was down-regulated in group G,MDA and ROS contents were significantly decreased,the SOD activity was increased,and the expression of PKCα,HO-1 and Mfn1 protein and mRNA was upregulated in group P (P＜0.05),and no significant change was found in the parameters mentioned above in group D (P＞0.05).Conclusion Promotion of Mfn1 expression following PKCα/HO-1 signaling pathway activation is the endogenous protective mechanism of endotoxin-induced damage to alveolar macrophages of rats.
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Objective@#To investigate the role of Mic60 in cardiac aging.@*Methods@#Wild-type and Mic60+ /- male mice at age of 4-6 months (young group, n=6) and 18-20 months (aged group, n=9) were used. H&E and Masson staining of frozen and paraffin sections were subjected to morphologic evaluation of the cardiac tissue samples. SA-β-Gal staining was utilized to detect the activity of senescence-associated β-galactosidase. Western blot was performed to detect the expression of Mic60 and p21 in cardiac tissues.@*Results@#Expression of Mic60 in mouse cardiac tissue increased in an age-dependent manner. Haploid insufficiency of Mic60 resulted in an increased left ventricular wall thickness [(1.32±0.09) mm vs.(1.12±0.09) mm, P<0.05], cardiomyocyte hypertrophy[(474.9±27.6) μm2 vs.(358.8±48.7) μm2, P<0.05] and interstitial fibrosis [ (38.24±7.58) ×103μm2 vs.(25.81±4.12)×103μm2, P<0.05], increased activity of SA-β-Gal (2.26±0.24 vs.0.25±0.05, P<0.01) and higher expression of p21 (P<0.01) in aged mouse cardiac tissue, but not in young mice.@*Conclusion@#Haploid insufficiency of Mic60 leads to cardiac hypertrophy, interstitial fibrosis, increased activity of SA-β-Gal and higher expression of p21 in aged cardiac tissue in mice, suggesting that Mic60 may prevent cardiac aging.
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Objective To evaluate the effect of hydrogen on the expression of Drp1 in the myocardial mitochondria of septic mice.Methods Fifty-four pathogen-free healthy male ICR mice,aged 6 weeks,weighing 20-25 g,were divided into 3 groups (n=18 each) using a random number table:sham operation group (S group),sepsis group (Sep group) and hydrogen group (H2 group).Sepsis was produced by cecal ligation and puncture in anesthetized mice.The mice inhaled 2％ hydrogen for 1 h starting from 1 and 6 h after operation in group H2.The mice were sacrificed at 1 day after operation,and hearts were removed for microscopic examination of the pathological changes which were scored and for determination of apoptosis in cardiomyocytes.Apoptosis rate was calculated.The myocardial mitochondria were isolated for measurement of respiratory control ratio (RCR) (using a Clark-type electrode),ATP content (by using a bioluminescence technique) and Drp1 expression (by Western blot).Results Compared with group S,the pathological score and apoptosis rate were significantly increased,the RCR and ATP content in mitochondria were decreased,and the expression of Drp1 was up-regulated in Sep and H2 groups (P＜0.05).Compared with group Sep,the pathological score and apoptosis rate were significantly decreased,the RCR and ATP content in mitochondria were increased,and the expression of Drp1 was down-regulated in group H2 (P＜0.05).Conclusion The mechanism by which hydrogen mitigates myocardial injury is related to down-regulating expression of Drp1 in the myocardial mitochondria and improving mitochondrial function in septic mice.
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Objective To evaluate the role of phosphatidylinositol 3-kinase/serine-threonine kinase (PI3K/Akt) signaling pathway in carbon monoxide (CO)-induced up-regulation of the mitofusin-1 (Mfn1) expression in endotoxin-challenged rat alveolar macrophages.Methods Alveolar macrophages obtained from the rats aged 12-20 weeks were subcuhured and seeded in 96 well plates at a density of 4× 104 cells/ml.After being cultured for 24 h,the cells were divided into 4 groups (n=10 each) using a random number table:control group (group C),endotoxin group (group L),lipopolysaccharide (LPS) +CO-releasing molecule-2 (CORM-2) group (group L+C) and LPS+CORM-2+PI3K inhibitor LY294002 group (group L+C+LY).Cells were cultured normally in group C.Cells were stimulated by using LPS 10 μg/ml in L,L+C and L+C+LY groups.In group L+C,CORM-2 100 μmol was given at 1 h before stimulation with LPS.In group L+C+LY,LY294002 20 μg and CORM-2 100 μ mol were given at 1.5 and 1.0 h before stimulation with LPS,respectively.The cells were continuously incubated for 24 h after the end of treatment.The concentrations of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in the supernatant were determined by enzyme-linked immunosorbent assay.The expression of PI3K,phosphorylated Akt (p-Akt) and Mfn1 in cells was measured by real-time polymerase chain reaction and Western blot.Results Compared with group C,the concentration of TNF-α was significantly increased,and the IL-10 concentration was decreased in L,L+C and L+C+LY groups (P＜0.05).Compared with group L,the concentration of IL-10 was significantly increased,the TNF-α concentration was decreased,and the expression of PI3K,p-Akt and Mfn1 was up-regulated in group L+C (P＜0.05).Compared with group L+C,the concentration of IL-10 was significantly decreased,the TNF-α concentration was increased,and the expression of PI3K,p-Akt and Mfn1 was down-regulated in group L+C+LY (P＜0.05).Conclusion PI3K/Akt signaling pathway is involved in CO-induced up-regulation of Mfn1 expression in endotoxin-challenged rat alveolar macrophages.
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Background Research comfirmed that second mitochondrial activator of caspase (Smac) is a promoting tumor cell apoptosis protein.Our previous study showed that the expression level of Smac in LECs is obviously higher in cataract than that in normal eyes.We assumed that silencing Smac gene in LECs can inhibit the apoptosis of LECs.The way to transfect Smac siRNA into LECs is a key step.Objective This study was to construct siRNA lentiviral vector of Smac and identify its silencing efficiency in human lens epithelial B3 cell line (HLE-B3) and establish low-expressed Smac HLE-B3 line.Methods Based on the genebank and our previous study,siRNA sequence of Smac was designed and composed.The synthetic double-stranded DNA was linked to the lentiviral vector GVll8 by T4 DNA ligase and then transformed DH5α competent cells.The plasmids were transformed into the DH5α competent cells.Recombinant colonies were screened by PCR and sequenced.Recombinant plasmids and two other auxiliary plasmids were used to infect 293T cells.Cell culture supernatant was collected for the measurement of viral titer.Recombinant lentiviral vector was used to infect HLE-B3 cells to calculate the viral multiplicity of infection (MOI) under the fluorescence microscope.Transfection efficiency was examined by calculating the GFP-positive cells.HLE-B3 cells were divided into negative control group,siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group.The relative expression levels of Smac mRNA in the cells were detected and compared among the three groups by real-time fluorescent quantitative PCR.Results GV118-Smac-siRNA was successfully constructed with the positive colonies 340 bp and blank vector colonies 299 bp,and viral titer was 3.0× 108 TU/ml.At a MOI of 100,the infecting efficiency of the vector on HLE-B3 cells was about 82％ and the cytotoxicity was low.The relative expression levels of Smac mRNA were (101.290±8.349)％,(92.330±6.320)％ and (32.540±4.221)％ in the negative control group,siRNA plasmid tranfected group and GV118-Smac-siRNA1 tranfected group,respectively,showing a significant difference among the three groups(F =32.871,P＜0.01),and the relative expression level of Smac mRNA was significantly lower in the GV118-Smac-siRNA1 tranfected group than that in the negative control group (P =0.000).However,no significant difference was found in the Smac mRNA expression between the blank plasmid group and the negative control group (P=0.535).Conclusions GV118-Smac-siRNA lentiviral vector is successfully constructed.Smac-siRNA can effectively inhibit the expression of Smac mRNA in human LECs.
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Objective To evaluate the role of mitochondrial fusion?fission in endotoxin?induced a?cute lung injury in rats. Methods Twenty healthy male Sprague?Dawley rats, weighing 160-180 g, were e?qually and randomly divided into either control group ( group C ) or endotoxin?induced acute lung injury group (group L) using a random number table. Lipopolysaccharide 5 mg∕kg was injected intravenously in group L, while the equal volume of normal saline 0?5 ml was given instead in group C. The animals were sacrificed at 6 h after administration of lipopolysaccharide or normal saline. The lungs were immediately re?moved for measurement of wet to dry lung weight ratio ( W∕D ratio) , superoxide dismutase ( SOD) activity and malondialdehyde ( MDA) content. The mitochondrial fusion proteins mitofusin 1 ( Mfn1) , Mfn2 and op?tic atrophy 1 ( OPA1) mRNA and protein expression was detected, and mitochondrial fission proteins dy?namin?related protein 1 (Drp1) and fission 1 (Fis1) mRNA and protein expression was also detected in lung tissues. Results Compared to group C, the W∕D ratio and MDA contents in lung tissues were signifi?cantly increased, SOD activity was decreased, Mfn1, Mfn2 and OPA1 mRNA and protein expression in lung tissues was down?regulated, and Drp1 and Fis1 mRNA and protein expression was up?regulated in group L. The pathological damage to lung tissues was obviously aggravated in group L when compared to group C. Conclusion The mechanism underlying endotoxin?induced acute lung injury is related to enhanced oxidative stress responses caused by decreased mitochondrial fusion and increased mitochondrial fission in rats.
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Objective To examine the complement component 1 Q subcomponent-binding protein (C1QBP) gene expression in human resistance choriocarcinoma cell lines and its parental cell line JeG-3,and to investigate whether silence C 1QBP by small interference RNA could reverse the resistance of human resistance choriocarcinoma cell lines to its relevant chemotherapy drugs.Methods Expression of C1QBP mRNA and protein in cells were detected by real-time fluorogenic quantitative PCR and western blot,respectively.The difference of C 1QBP expression was compared between human resistance choriocarcinoma cell lines and its parental cell line JeG-3.Sub-cellular location was proved by confocal immunofluorescence microscopy.A lentiviral vector containing short hairpin RNA (shRNA) targeting C 1QBP was constructed and cotransfected with the packaging plasmid mixture into 293T cells by lipofectamine 2000.The human resistance choriocarcinoma cell lines were infected with the packaged lentivirus.Real-time fluorogenic quantitative PCR and western blot were used to validate whether the C 1QBP gene expression was silenced.The cell counting kit 8(CCK8)was used to determine the drug sensitivity.Results (1)The C1QBP mRNA expression levels among four human resistance choriocarcinoma cell lines[JeG-3/floxuridiuum (FUDR),JeG-3/methotrexate (MTX),JeG-3/etoposide (VP),JeG-3/dactinomycin (KSM)] were 2.520±0.680,1.770±0.230,1.940±0.090 and 1.740±0.350 folds compared to that in JeG-3 cells.The C1QBP protein was higher expression level in human resistance choriocarcinoma cell lines than that in JeG-3.The immunofluorescence methods and confocal analysis showed that C1QBP localized predominantly in the mitochondrial matrix.(2)The C1QBP mRNA expression in JeG-3/FUDR cells after infected with lentiviral vector were decreased by 93.1％ (P＜0.01).The protein expression of C 1QBP in JeG-3/FUDR cells after infected with lentiviral vector were almost completely suppressed.The resistance indexes of four human resistance choriocarcinoma cell lines(JeG-3/FUDR,JeG-3/MTX,JeG-3/VP,JeG-3/KSM) were respectively 86.3％,93.9％,92.8％ and 89.9％,which were decreased remarkably by knockdown the C 1QBP expression (P＜0.05).Conclusions C1QBP is overexpressed in human resistance choriocarcinoma cell lines compared with parental cell line JeG-3.Inhibition of C 1QBP by lentivirus-mediated small interference RNA could effectively reverses the resistance of human resistance choriocarcinoma cell lines to its relevant chemotherapy drugs.
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ObjectiveTo report clinical, pathological and molecular genetic features in a Chinese family with hereditary motor and sensory neuropathy type 6. MethodsThe index case is a 15 years old boy.He developed progressive distal limb weakness at the age of 5.The disease deteriorated slowly,accompanied with contracture of achilles' tendon. At the age of 11 years old he suffered from decrease of visual acuity. At the age of 12, he found the muscular atrophy of both hands without sensory disturbances.Visual evoked potential revealed prolonged latency of bilateral P100. Ophthalmological examination showed bilateral optic atrophy. His mother had the similar symptoms at the age of 7 and reduced visual acuity at the age of 10. Nerve conduction velocity revealed in both pat1ents no compound motor and sensory nerve action potentials in most nerves or slightly reduced nerve conduction velocities with severely reduced amplitudes of the compound motor and sensory nerve action potentials. Sural nerve biopsy was performed on the proband.The sequence of MFN2 gene was analyzed in DNA from the index, his mother and 100 healthy controls.ResultsSural nerve biopsy revealed severe loss of myelinated fibers with few regenerating clusters.Ultrapathological examination showed a few of atypical bulbs of myelinated fibers, occasionally regenerating clusters, mitochondrial swelling and aggregation in a few of axons. A new mutation of W740R mutation in MFN2 gene has been identified in the index case, her mother, but not in 50 healthy controls. Conclusions A novel MFN2 gene mutation result in hereditary motor and sensory neuropathy type 6.Mild visual loss appeares after the lesion of spinal nerves. Demyelination of peripheral nerve appears in the disease.
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ObjectiveThe purpose of this study was to investigate the clinical significance of HMGB1 and Smac/DIABLO protein expression in epithelial ovarian cancer.MethodsImmunohistochemistry (IHC) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining were used to detect the protein expression of HMGB1,Smac/DIABLO and cell apoptosis in 45 epithelial ovarian cancers by tissue microarray.ResultsThe protein expression of HMGB1 in epithelial ovarian cancers was significant higher than benign cancers and normal ovarian tissues( P ＜0.05).The protein expression of HMGB1 was related to the lymph node metastasis,which had a negative correlation with the cell apoptosis index ( rs =-0.583,P =0.000).The protein expression of Smac/DIABLO in epithelial ovarian cancers was significantly lower than benign cancers and normal ovarian tissues( P ＜0.05) ; the protein expression of HMGB1 in epithelial ovarian cancers had a negative relationship with protein expression of Smac/DIABLO( rs =-0.40,P =0.006).ConclusionsThe over-expression of HMGB1 in epithelial ovarian cancers may play an important part in the metastasis of the epithelial ovarian cancers.The over-expression of HMGB1 and the low-expression of Smac/DIABLO may take part in the occurrence and development of the epithelial ovarian cancers through abnormal cell apoptosis.
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Objective To investigate the effects of high-fat diets and rosiglitazone treatment on the expressions of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and mitofusin-2 (Mfn2) in skeletal muscle of aged rats. Methods Male wistar rats aged 21-23 months were randomly divided into old control group (OC; n=20), high-fat diets group (HF; n=20) and high-fat diets plus rosiglitazone treatment group (RSG; n=20). Male wistar rats aged 4-5 months were selected as young control group (YC; n=20). Insulin sensitivity was evaluated by hyperinsulinemic-euglycemic clamp technique at the end of the 4th and 8th week. The expressions at mRNA and protein levels of PGC-1α and Mfn2 in skeletal muscle were determined by polymerase chain reaction and Western blotting technique. Results (1)After 8 weeks, the levels of free fatty acid [(0. 68±0. 18)mmol/L, (0.82±0. 23) mmol/L and (1. 53±0.40) mmol/L], triglyceride [(0.53±0.13) mmol/L,(0. 63±0. 17) mmol/L and (1.08±0.30) mmol/L]and muscle triglyceride [(1.09±0.17) mmol/L,(1.34±0. 20) mmol/L and (2.07±0. 30) mmol/L]in YC group, OC group and HF group were significantly increased and glucose infusion rate [(30.4±4. 2) mg·kg~(-1)·min~(-1), (20.9±2.2) mg·kg~(-1)·min~(-1) and (12. 0±1.9) mg·kg~(-1)·min~(-1)]was significantly decreased as compared with pre-high fat diet, respectively. The levels of fasting free fatty acid [(0.93±0.29) mmol/L], triglyceride [(0.62±0.12) mmol/L]and music triglyceride [(1.68±0.28) mmol/L]in RSG group were significantly decreased and glucose infusion rate [(16.7±1.7) mg·kg~(-1)·min~(-1)]was significantly higher than in HF group. (2)In skeletal muscle, the expressions at mRNA and protein levels of PGC-1α and Mfn2 decreased in OC group compared with YC group (all P<0.01). The expressions of PGC-1α and Mfn2 were lower significantly in HF group than in OC group, and were higher significantly in RSG group than in HF group (all P<0.01). Conclusions The aged rats fed the high-fat diets develop insulin resistance with decreased expressions of PGC-1α and Mfn2 in skeletal muscle. Insulin sensitivity is improved with rosiglitazone treatment by increasing expressions of PGC-1α and Mfn2 in skeletal muscle.
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Objective To report clinical and pathological features in a Chinese family with CharcotMarie-Tooth disease type 2A2 (CMT2A2). Methods There were 5 patients (2 male and 3 female) in a family with an autosomal dominant inheritance pattern. The index case was a 36 years old woman. She developed progressive distal limb weakness at the age of 6, with pescavus at the age of 8. Other 4 family members presented with similar symptoms between the age of 3-7. Physical examination showed distal limb weakness and wasting, loss of sensory and contracture in all of them. Nerve conduction velocity revealed non-potential in several motor and sensory nerves in the proband and her son. Sural nerve biopsy was performed on the proband. The sequence of MFN2 gene was analyzed in DNA from 5 patients and 3 asymptomatic members. Results Sural nerve biopsy revealed severe loss of myelinated fibers with few regenerating clusters. Electron microscopy revealed aggregation of mitochondrian in the axons. A R94W mutation in MFN2 gene has been identified in 5 patients, but not in unaffected members. Conclusions We confirmed Chinese CMT2A2. Absent of regenerating cluster in the nerve indicated that MFN2 mutation predominantly resulted in lesions in the neurons.
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Objective To investigate the expression of XIAP, Smac, HtrA2 and XAF1 in the hippocampus following SE in rats and to explore the pathophysiological mechanisms of expression of XIAP and its negative regulators after SE. Methods The lithium-pilocapine model of status epilepticus was established in SD rat. XIAP, Smac, HtrA2, XAF1 and activated caspase-3 protein were examined using immunohistochemistry. Western blot was used to detect the protein levels of XIAP, Smac, HtrA2 and activated easpase-3. Results XIAP immunoreactivity diffusely distributed within the neuron after SE. Compared with the control group, the expression of CA3 XIAP protein in the SE group was increased gradually since 2 hours (0.5503±0.0172 vs 0.1507±0.0165, t=115.87, P<0.01), peaking at 8 hours (0.6221±0.0238 vs 0.1507±0.0165, t=136.69, P<0.01). The expression of CA3 Smac, HtrA2, XAF1 and activated caspase-3 protein were increased generally following SE. Western blot analysis showed a significant increase in Stoat, HtrA2, activated caspase-3 protein levels from 2 to 72 hours following SE, but no significant differences were seen in XIAP protein levels between the control group and the SE group. Conclusions The XIAP, Smac, HtrA2 and XAF1 are involved in the regulation of neuronal apoptosis and implicated in pathophysiological mechanisms of neuronal damage after SE.