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Objective:To study the effect and mechanism of angiotensin type 1 receptor (AGTR1) blocker olmesartan (OMS) on the apoptosis of human Tenon capsule fibroblasts (HTF).Methods:Tenon capsule tissues were obtained from patients during strabismus surgery in the Second Affiliated Hospital of Xi'an Jiaotong University.Primary HTF were cultured by explant culture.Primary cells were identified by vimentin immunofluorescence staining and flow cytometry.The fibrosis model of HTF was established using 10 ng/ml transforming growth factor-β2 (TGF-β2). The cells were divided into normal control group cultured in culture medium, TGF-β2 group in culture medium containing TGF-β2, TGF-β2+ OMS group in culture medium containing TGF-β2 and OMS, and OMS group in culture medium containing OMS, and were cultured for 48 hours.Cell apoptosis was detected by flow cytometry with annexin V/PI staining.The early apoptosis, late apoptosis, and total apoptosis rates were analyzed.The protein expression of procaspase-9, cleaved caspase-9, bax and bcl-2 in the mitochondrial apoptosis pathway was detected by Western blot.The activity of lactate dehydrogenase (LDH) and superoxide dismutase (SOD) was detected by colorimetry.The study protocol was approved by the Ethics Committee of the Second Affiliated Hospital of Xi'an Jiaotong University (No.2019-014).Results:Primary HTF were successfully isolated and cultured.The cultured cells were long spindle-shaped and positive for vimentin.The expression rate of vimentin in the primary cells was greater than 99%.A statistically statistical difference was found in the early apoptosis rate, late apoptosis rate, and total apoptosis rate among the four groups ( F=24.92, 3.96, 41.82; all at P<0.05). The early and total apoptosis rates were significantly higher in TGF-β2+ OMS group than normal control group and TGF-β2 group, and the late apoptosis rate in TGF-β2+ OMS group was significantly higher than that of normal control group (all at P<0.05). There were statistically significant differences in cleaved caspase-9/procaspase-9, bax, and bax/bcl-2 among the four groups ( F=4.40, 7.98, 4.61; all at P<0.05). The bax/bcl-2 expression was significantly increased in TGF-β2+ OMS group in comparison with normal control group, and the expressions of cleaved caspase-9/procaspase-9, bax, and bax/bcl-2 were significantly elevated in TGF-β2+ OMS group compared with TGF-β2 group (all at P<0.05). LDH activity in the normal control group, TGF-β2 group, TGF-β2+ OMS group and OMS group was (783.99±79.97), (913.16±196.86), (2 529.06±240.21), and (2 134.29±138.96) μmol/(min·L), respectively, showing a statistically significant difference ( F=24.95, P<0.05). Compared with normal control group and TGF-β2 group, LDH activity in TGF-β2+ OMS group was increased, and the differences were statistically significant (both at P<0.05). SOD activity in the normal control group, TGF-β2 group, TGF-β2+ OMS group and OMS group was (50.35±0.97), (41.61±4.56), (28.88±3.26), and (37.61±4.83) μmol/(min·L), respectively, showing a statistically significant difference ( F=5.71, P<0.05). SOD activity was reduced in TGF-β2+ OMS group compared with normal control group and TGF-β2 group, reduced in OMS group compared with normal control group, and the differences were statistically significant (all at P<0.05). Conclusions:AGTR1 blocker OMS can promote the apoptosis of HTF effectively.Mitochondrial apoptosis pathway mediated by bax/bcl-2/caspase-9 and oxidative stress pathway are the potential mechanisms that OMS regulates the apoptosis of HTF.
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Objective:To investigate the effect of the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3 (NLRP3) inflammasome on the proliferation and apoptosis of human retinal pigment epithelial cell line ARPE-19 exposed to high glucose and its mechanism.Methods:ARPE-19 cells cultured in vitro were divided into normal control group and high-glucose group, and were cultured in conventional medium and medium containing 30 mmol/L glucose for 48 hours, respectively.The content of reactive oxygen species (ROS) were detected by fluorescent probe, and the activity of superoxide dismutase (SOD) and the concentration of malondialdehyde (MDA) were tested by biochemical assay.The cells of the two groups were cultured with 0, 2, 5, 10, 15 and 20 μmol/L NLRP3 inhibitor CY-09 for 48 hours, respectively.The proliferation rate of ARPE-19 cells under various concentrations of CY-09 treatment was detected by cell counting kit-8, and the appropriate concentration of CY-09 was determined.ARPE-19 cells were divided into normal control group, normal+ CY-09 group, high-glucose group and high glucose+ CY-09 group.The culture medium in the normal+ CY-09 group and high glucose+ CY-09 group was supplemented with 15 μmol/L CY-09.Flow cytometry was used to detect the apoptosis rate of each group, and Western blot was used to detect the relative expression levels of NLRP3, apoptosis-associated point protein (ASC), Caspase-1 precursor (pro-Caspase-1) and active fragments (cleaved-Caspase-1), B lymphocytoma-2 protein (Bcl-2), Bcl-2-associated X protein (Bax), Caspase-3 precursor (pro-Caspase-3) and active fragments (cleaved-Caspase-3). Results:The intensity of ROS fluorescence and MDA concentration were 120 020±3 245, (4.92±0.09) nmol/mg in the high-glucose group, which were both significantly higher than 35 426±811 and (1.78±0.03) nmol/mg in the normal control group, and the SOD activity was (35.65±1.22) μmol/(min·mg) in the high-glucose group, which was significantly lower than (74.96±1.41) μmol/(min·mg) in the normal control group, showing statistically significant differences between the two groups ( t=35.760, 46.960, 29.830; all at P<0.05). The proliferation rate of RPE cells in high-glucose group was significantly lower than that in normal control group, and the difference was statistically significant ( t=18.820, P<0.05). With the increase of CY-09 concentration, the proliferation rate of cells in the high-glucose group was gradually increased.The proliferation rates of cells treated with 10, 15 and 20 μmol/L CY-09 were all significantly higher than those treated with 0 μmol/L CY-09, showing statistically significant differences between them (all at P<0.05). The proliferation rates of cells treated with 15 μmol/L and 0 μmol/L CY-09 were not significantly different in the normal control group ( P>0.05). The apoptosis rate of cells in the high-glucose group was (21.68±0.41)%, which was significantly higher than (6.67±1.05)% in the normal control group and (13.96±0.07)% in the high-glucose+ CY-09 group, and the differences were statistically significant (both at P<0.05). The relative expression levels of NLRP3, ASC, cleaved-Caspase-1, cleaved-Caspase-3 and Bax proteins were significantly higher and the relative expression levels of Bcl-2 protein was significantly lower in the high-glucose group compared with the normal control group, and the differences were statistically significant (all at P<0.05). The relative expression levels of NLRP3, ASC, the active fragment of cleaved-Caspase-1, Bax and cleaved-Caspase-3 proteins were decreased and the relative expression levels of Bcl-2 protein were increased in the normal+ CY-09 group and high glucose+ CY-09 group compared with the normal control group and high glucose group, and the differences were statistically significant (all at P<0.05). Conclusions:NLRP3 inflammasome mediates the high glucose induced RPE cells apoptosis through ROS/NLRP3/Caspase-1 signaling pathway.
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OBJECTIVE:To investigate the effects of loading-dose rosuvastain before early percutaneous coronary intervention (PCI) on reperfusion arrhythmias in patients with acute non-ST-segment elevation myocardial infarction (NSTEMI).METHODS:A total of 136 patients with NSTEMI were randomly divided into loading-dose group (68 cases) and control group (68 cases).Both groups who were not given anti-platelet drugs were given loading-dose of Aspirin enteric-coated tablets 300 mg+Clopidogrel sulfate tablets 600 mg immediately after admission.The patients who were given aspirin regularly were given loading-dose of Clopidogrel sulfate tablets 600 mg only once after admission.The patients who were given clopidogrel regularly were given loading-dose of Aspirin enteric-coated tablets 300 mg only once.Those received PCI 12-24 h after medication.After PCI,they took Aspirin enteric-coated tablets 100 mg for life+Clopidogrel bisulfate tablets 75 mg at least 12 months.Loading-dose group was given loading-dose of Rosuvastatin calcium tablets 20 mg orally,12 h before surgery.All patients began to take Rosuvastatin calcium tablets 10 mg,once a day,since the night after the operation.Coronary angiography and the occurrence of reperfusion arrhythmia were observed in 2 groups.The levels of CK-MB and cTnT,major adverse cardiovascular events (MACE) were observed before and after surgery.RESULTS:There was no statistical significance in the number of diseased vessels,culprit vessels,the degree of culprit vessels stenosis or the incidence of MACE between 2 groups (P＞ 0.05).The incidence of reperfusion arrhythmia in loading-dose group was significantly lower than control group,with statistical significance (P＜0.01).There was no statistical significance in the degree of culprit vessels stenosis between 2 groups (P＞0.05).Before surgery,there was no statistical significance in the levels of CK-MB or cTnT between 2 groups (P＞0.05).After surgery,the levels of CK-MB and cTnT in 2 groups were significantly higher than before surgery,but the loading-dose group was significantly lower than the control group,with statistical significance (P＜0.01).CONCLUSIONS:Preoperative loading-dose of rosuvastatin before PCI can reduce the incidence of reperfusion arrhythmias in NSTEMI patients.
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Objective: To investigate the effects of domestic bivalirudin on plasma intercellular adhesion molecular-1 (ICAM-1) during percutaneous coronary intervention (PCI).Methods: Sixty PCI candidates were randomly divided into heparin group (n =30) and bivalirudin group (n =30).They were respectively treated with intravenous heparin and domestic bivalirudin as the anticoagulants during PCI.ICAM-1 in blood was measured before PCI and in 2h, 1d and 7d after PCI, respectively.Results: In heparin group, ICAM-1 level decreased significantly in 2 h after the intravenous injection when compared with that before the injection and that in bivalirudin group at the same time point (P＜0.05).No significant differences in ICAM-1 level were found on the 1st and 7th day after PCI in the two groups when compared with that before the administration (P＞0.05).Mild bleeding occurred in three patients receiving heparin and one patient receiving bivalirudin,but there was no significant difference after PCI.Conclusion: Compared with bivalivudin, heparin has a short inhibitory effect on the expression of ICAM-1 during PCI.It is beneficial to the patients with kidney disease or stroke during PCI.
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OBJECTIVE:To evaluate the effects of loading-dose rosuvastatin on vascular endothelial function in patients with non-ST segment elevation acute coronary syndromes (NSTE-ACS) after early interventional therapy. METHODS:Totally of 128 NSTE-ACS patients underwent early interventional therapy were randomly divided into conventional dose group (63 cases) and loading dose group(65 cases). Before operation,all patients were given Clopidogrel sulfate tablets 300 mg and Aspirin enteric-coat-ed tablets 100 mg;on this basis,conventional dose group was given Rosuvastatin calcium tablets 10 mg orally;loading dose group was given Rosuvastatin calcium tablets 20 mg orally. After PCI,both groups were given Rosuvastatin calcium tablets 10 mg orally, qd,and Aspirin enteric-coated tablets(100 mg/d)and Clopidogrel sulfate tablets(75 mg/d),for consecutive 3 months. The blood samples were collected before surgery,8 h and 24 h after surgery. The serum levels of CK-MB,cTn T,hs-CRP,ET and NO were detected. The occurrence of major adverse cardiovascular events was recorded within 3 months after surgery. RESULTS:Compared with before surgery,the serum levels of CK-MB,cTn T,hs-CRP and ET were increased significantly 8 h and 24 h after surgery, while the level of NO was decreased,with statistical significance (P0.05). CONCLUSIONS:For NSTE-ACS patients underwent ear-ly interventional therapy,loading dose of rosuvastatin can protect the patients and inhibit the injury of vascular endothelial cell in-duced by the surgery.
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Objective The purpose of this study is to determine whether atrial expression of hepatocyte growth factor (HGF), connective tissue growth factor (CTGF) and transforming growth factor-beta1 (TGF-13,) is altered in patients with rheumatic heart disease (RHD) and chronic atrial fibrillation (AF).Methods Atrial tissue was obtained from the right atrial appendage during heart surgery from 35 patients. In 27 patients with rheumatic heart disease, 8 patients had no history of AF, and 19 had chronic persistent AF (≥6 months cAF). Other 8 patients with congenital heart disease had no history of AF were the control group.The mRNA expression of HGF, CTGF, TGF-β1, collagen Ⅰ and collagen Ⅲ was measured by the real-time fluorescence quota polymerase chain reaction (real time PCR). The fibrosis of right atrial appendage was detected by HE and Masson staining. Results The amount of CTGF, TGF-β1, collagen Ⅰ and collagen Ⅲ was significantly increased in patients with sinus rhythm compared with the control group (P<0.05) and further increased in patients with chronic AF compared with patients with sinus rhythm (P<0.05). The amount of HGF was significantly decreased in patients with chronic AF compared with patients with sinus rhythm and the control group (P<0.05). But the difference of the latter two groups was not statistically significant.Correlation analysis demonstrated that the mRNA expression of collagen Ⅰ , collagen Ⅲ, TGF-β1, and CTGF in right atrial appendage of RHD and AF was positively correlated with the left atrial diameter and the area of myocardial fibrosis. Conclusion In human atrium with RHD, the mRNA expression of collagen Ⅰ , collagen Ⅲ, CTGF and TGF-β1, is up-regulated. HGF has anti-fibrosis function and the down-regulation of its mRNA expression in patients with RHD may be an important factor in the initiation or maintenance of AF.