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Objective:To investigate the regulatory effects of endogenous nitric oxide (NO) on the activity of superoxide dismutase-1 (SOD1) and apoptosis of human umbilical vein endothelial cells (HUVECs).Methods:HUVECs were taken as the research object.The endothelial NO synthase (eNOS) short hairpin RNA(shRNA) lentivirus was employed to transfect HUVECs to knock down eNOS.HUVECs were divided in 4 groups: the scramble group, the eNOS shRNA group, the eNOS shRNA + sodium nitroprusside(SNP) group and the eNOS shRNA+ SNP+ tris (2-carboxyethyl) phosphine hydrochloride (TCEP) group.The protein expressions of eNOS and SOD1 dimer/monomer in cells were detected by western blot.The activity of SOD was detected by the enzyme-linked immunosorbent assay.The NO content in cells was detected with NO fluorescence probe.The level of superoxide anion in HUVECs was detected with dihydropyridine (DHE). The terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay was adopted to detect the apoptosis of HUVECs in situ.Results:Compared with the scramble group, the endogenous NO content (2.690±0.420 vs.15.029±2.193, P<0.01), eNOS protein expression (1.000±0.778 vs.3.141±0.199, P<0.01), SOD1 dimer/monomer ratio (4.6±1.0 vs.7.6±2.0, P<0.05) and SOD activity [(0.432±0.254) Carmen′s unit/10 4 cell vs.(1.000±0.116) Carmen′s unit/10 4 cell, P<0.01] were significantly decreased, while the level of intracellular superoxide anion (11.180±1.560 vs.6.146±1.007, P<0.01) and HUVECs apoptosis [75.0 (55.0, 100.0)% vs.0 (0, 0)%, P<0.01] were significantly increased in the eNOS shRNA group.Compared with the eNOS shRNA group, the content of endogenous NO (16.705±0.116 vs.2.690±0.420, P<0.01), the ratio of SOD1 dimer/monomer (7.3±2.0 vs.4.6±1.0, P<0.05) and the activity of SOD [(0.737±0.060) Carmen′s unit/10 4 cell vs.(0.432±0.254) Carmen′s unit/10 4 cell, P<0.05] were significantly increased, while the level of superoxide anion (6.897±1.648 vs.11.180±1.560, P<0.01) and the HUVECs apoptosis [0 (0, 0)% vs.75.0 (55.0, 100.0)%, P<0.01] were significantly decreased in the eNOS shRNA+ SNP group.Compared with the eNOS shRNA + SNP group, the ratio of SOD1 dimer/monomer (4.4±0.9 vs.7.3±2.0, P<0.05) and the activity of SOD [(0.214±0.084) Carmen′s unit/10 4 cell vs.(0.737±0.060) Carmen′s unit/10 4 cell, P<0.01] were significantly decreased, while the level of superoxide anion (10.917±1.552 vs.6.897±1.640, P<0.01) and the apoptosis level of HUVECs[63.6 (55.0, 90.0)% vs.0 (0, 0)%, P<0.01] were significantly increased in the eNOS shRNA+ SNP+ TCEP group.However, there was no significant difference in the NO content (16.112±0.926 vs.16.705±0.116, P>0.05). Conclusions:Endogenous NO could effectively antagonize the apoptosis of endothelial cells by increasing the cysteine-dependent SOD1 dimer/monomer ratio, enhancing SOD activity and inhibiting the accumulation of reactive oxygen species.
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Objective To explore the effect of different concentrations of endothelin-1 (ET-1)on the en-dogenous nitric oxide (NO)and hydrogen sulfide (H2S)pathways of vascular smooth muscle cells (A7r5 cell lines)in rats.Methods A7r5 cell lines were divided into the control group and the experimental group.ET-1 at a concentra-tion of 10 -8-10 -6 mol/L was added into the experimental group,and as for the control group,the same volume of sterile phosphate buffered saline (PBS)buffer solution was added.The content of NO and H2S in A7r5 cell lines was detected by fluorescent NO probe and H2S probe after ET-1 stimulation for 48 h,respectively.The content of NO in the supernatant was measured by NO assay kit at 48 h of the incubation.The content of H2S in the supernatant was measured by polarographic H2S sensor at 48 h of the incubation. The expressions of inducible nitric oxide synthase (NOS2),endothelial nitric oxide synthase (NOS3),cystathionine -γ -lyase (CSE),cystathionine -β -synthase (CBS)and proliferating cell nuclear antigen (PCNA)were detected by the Western blot method.Results The rela-tive fluorescence intensity of the content of NO in the A7r5 cell lines of ET-1 10 -8,10 -7 and 10 -6mol/L groups (0. 078 ± 0. 080,0.075 ± 0.002,0.056 ± 0.009)was markedly lower than that in the control group(0.094 ± 0. 061), and the differences were statistically significant(F＝15.248,P＜0.05);Compared with the control group[(2. 131 ± 0. 484)μmol/L],the content of NO in the supernatant of the experimental groups [(1.391 ± 0.134 )μmol/L, (1.219 ± 0. 280)μmol/L,(1.116 ± 0.181)μmol/L]was significantly decreased,and the differences were statistically significant(F＝20.833,P＜0.01);NOS2 protein expression(0.457 ± 0.097,0.462 ± 0.116,0.438 ± 0.180)was decreased markedly compared with that of the control group(0.721 ± 0.222),and the differences were statistically sig-nificant(F＝6.196,P＜0.01),but the expression of NOS3 showed no significant differences(F＝2.669,P＞0.05). The relative fluorescence intensity of the content of H2S in the A7r5 cell lines of ET-1 10 -8,10 -7 and 10 -6mol/L groups (0.063 ± 0.002,0.056 ± 0.008,0.042 ± 0.009)was markedly lower than that in the control group (0.082 ± 0. 006),and the differences were statistically significant(F ＝16.297,P＜0.01);Compared with the control group [(29.439 ±4.236)μmol/L],the content of H2S in the supernatant of the experimental groups [(17.516 ±5.144) μmol/L,(14.481 ± 4.885)μmol/L]was significantly decreased,and the differences were statistically significant (F＝12.518,P ＜0.01).CBS protein expression(0.359 ± 0.096,0.270 ± 0.038,0.174 ± 0.051)was decreased markedly compared with that of the control group(0.707 ± 0.107),and the differences were statistically significant (F＝20.833,P＜0.01),and the expression of CSE showed no significant differences(F＝0.708,P＞0.05).The data showed that PCNA protein expression in the 10 -7mol/L ET-1 group(0.686 ± 0.180)significantly increased com-pared with that of the control group(0.437 ± 0.191),and the difference was statistically significant (t＝ -2.840,P＜0.01).Conclusion ET-1 stimulation can lead to the proliferation of vascular smooth muscle cells and down-regu-late its endogenous NO and H2S pathways.
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Objective To explore the effect of endogenous sulfur dioxide (SO2)on the apoptosis induced by cobalt chloride (CoCl2)in the human pulmonary arterial endothelial cells (HPAECs).Methods CoCl2was used in the primary HPAECs to mimize hypoxia-induced cell apoptosis.The aspartate aminotransferase 1(AAT1),and the key enzyme generating endogenous SO2 were over -expressed by transfecting HPAECs with lentivirus containing AAT1 cDNA.HPAECs were divided into 4 groups:vehicle group,vehicle + CoCl2 group,AAT1 group and AAT1 + CoCl2 group.The expressions of AAT1,B-cell lymphoma-2 (bcl-2),bcl-associated X protein (bax),Caspase-3 and activated Caspase-3 (cleaved Caspase-3)in the HPAECs were measured by Western blot.The AAT activity was assessed with colorimetry method.The SO2 content in the HPAECs was in situ observed by SO2-specific fluorescent probe.The HPAECs apoptosis was investigated by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)assay.Results There were significant differences in the endogenous SO2 content,the expre-ssions of AAT1 and bcl-2,and the ratio of cleaved Caspase-3/Caspase-3 among 4 groups of HPAECs were (F＝147.364,23.738,6.521,64.884,all P＜0.05).However,there was no difference in the expression of bax among 4 groups of HPAECs (F＝1.620,P＞0.05).Compared with vehicle group,AAT activity [(0.96 ± 0.24)Carmen's unit/μg vs.(2.21 ± 0. 60)Carmen's unit/μg],endogenous SO2 content (40.71 ± 7.72 vs.105.60 ± 16.20)and bcl-2 expression (0.59 ± 0.19 vs.1.02 ± 0.20)in the HPAECs of vehicle +CoCl2 group were significantly de-creased,while the cell apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (1.56 ± 0.25 vs.0.95 ± 0.13)were significantly increased (all P＜0.05).However,there were no differences in the expression of AAT1 (0. 50 ± 0.12 vs.0.53 ± 0.11)in the HPAECs between vehicle group and vehicle+CoCl2 group (P＞0.05). The SO2 content (351.50 ± 42.43 vs.105.60 ± 16.20)and AAT1 expression (1.22 ± 0.33 vs.0.53 ± 0.11)in the HPAECs of AAT1 group were higher than those of vehicle group (all P ＜0. 05 ). Compared with AAT1 group, endogenous SO2content (333.50 ± 46.22 vs.351.50 ± 42.43)and the expression of AAT1 (1.26 ± 0.36 vs.1.22 ± 0.33)and bcl-2 (1.14 ± 0.38 vs.1.03 ± 0.27)in the HPAECs of AAT1 +CoCl2group did not change (all P＞0. 05).Moreover,no difference was observed in the HPAECs apoptosis assessed by TUNEL and the ratio of cleaved Caspase-3/Caspase-3 (0.51 ± 0.17 vs.0.50 ± 0.11)between the two AAT1 -overexpressed groups (all P ＞0. 05).Conclusion Endo-genous SO2inhibited the hypoxic HPAECs apoptosis stimulated by the treatment of CoCl2.
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Objective To explore the changes in the endogenous sulfur dioxide (SO2) pathway in the myocardial hypertrophy induced by the angiotensin Ⅱ (Ang Ⅱ) in mice.Methods Fourteen healthy C57BL mice,9 weeks old,were randomly divided into control group(n =7) and Ang Ⅱ group(n =7),and capsule osmotic pump with pre loaded 9 g/L saline and Ang Ⅱ was implanted into the back of each mouse subcutaneously.Mter 2 weeks,the mice were executed.The heart weight/body weight (HW/BW) and the left heart weight/full heart weight (LVW/HW) of the mice were measured.The microstructure of the cardiac myocyte was observed by hematoxylin-eosin (HE) staining under the microscope.The expression of myocardial alpha myosin heavy chain (α-MHC) was detected by immunohistochemistry and Western blot methods.SO2 enzymes aspartate aminotransferase 1 (AAT1) and AAT2 protein expression were detected by Western blot method.Myocardial SO2 content and AAT activity were measured by high performance liquid chromatography with fluorimetric detection and colometric method.Results Compared with control group,the HW/BW and LVW/HW in mice of Ang Ⅱ group were significantly increased (all P ＜ 0.O1),the cardiac myocytes were hypertrophy,and α-MHC positive staining in the cytoplasm of myocardium was weakened.Moreover,Western blot data showed that α-MHC protein expression in heart tissue of Ang Ⅱ-treated mice was decreased significantly (allP ＜ 0.05).Simultaneously,the data showed that AAT2 protein expression,SO2 content and AAT activity in heart tissue of Ang Ⅱ-treated mice were also decreased markedly[(1.093 ±0.131) μ mol/g protein vs.(0.737 ±0.233) μmol/g protein,P ＜ 0.05;(7.979 ± 1.317) U/rmg protein vs.(6.470 ± O.516) U/mg protein,P ＜ 0.01].Furthermore,there was a negative correlation between LVW/HW and cardiac SO2 content in heart tissue (r =-0.56,P ＜ 0.05).Conclusions Myocardial endogenous SO2/AAT2 pathway is down-regulated in the development of myocardial hypertrophy induced by Ang Ⅱ in mice.
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Objective To investigate the regulatory effects of endogenous sulfur dioxide (SO2) on collagen accumulation in pulmonary arterial fibroblasts of rats and its mechanisms.Methods Primary rat pulmonary artery fibroblasts were used in the experiment and were divided into 3 groups:the control group,the L-aspartate-beta-hydroxamate(HDX) group and the HDX ± SO2 group.SO2 content of pulmonary artery fibroblasts supernatant was detected by adopting high performance liquid chromatography (HPLC).Collagen type Ⅰ and collagen type Ⅲ in pulmonary artery fibroblasts were determined by using immunofluorescence.Phosphorylation of Smad2/3,protein expression of matrix metalloproteinase (MMP)-13 and tissue inhibitors of MMP (TIMP)-1 were detected by using Western blot.One-way ANOVA was used for multiple group comparisons followed by Bonferroni test for each group.P ＜ 0.05 was considered as significant difference.Results Compared with control group,endogenous SO2 content in HDX group was significantly decreased [(14.30-± 0.48) μmol/L vs.(20.14 ± 0.49) μμmol/L,P ＜ 0.01],the level of Smad2/3 increased (1.03 ±0.31 vs.0.48 ± 0.20,P ＜ 0.01),protein expressions of MMP-13 and TIMP-1 in pulmonary artery fibroblasts were decreased (MMP-13:0.28 ± 0.06 vs.0.75 ± 0.11,P ＜ 0.01;TIMP-1:0.40 ± 0.05 vs.0.66 ± 0.20,P ＜ 0.01),and the ratio of MMP-13/TIMP-1 was decreased (0.71 ± 0.12 vs.1.23 ± 0.45,P ＜0.01).However,contents of collagen Ⅰ and collagen Ⅲ were significantly increased.Compared with HDX group,the level of Smad2/3 phosphorylation in HDX ± SO2 group decreased (0.57 ± 0.16 vs.1.03 ± 0.31,P ＜ 0.01),protein expression of MMP-13 and TIMP-1 upregulated (MMP-13:0.63 ± 0.06 vs.0.28 ± 0.06,P ＜ 0.01;0.59 ± 0.11 vs.0.40 ± 0.05,P =0.015),the ratio of M MP-13/TIMP-1 (1.10 ± 0.22 vs.0.71 ± 0.12,P =0.033) increased,but contents of collagen type Ⅰ and type Ⅲ were reduced obviously.Conclusions SO2 promotes the degradation of collagen and collagen accumulation in pulmonary artery fibroblasts of rats probably by inhibiting Smad2/3 signal pathway,increasing protein expression of MMP-13 and TIMP-1,and upregulating the ratio of MMP-13/TIMP-1.
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Objective To observe the effect of sulfur dioxide (SO2) on vasoactive peptides in aorta of atherosclerotic(AS) rats.Methods Twenty-eight male SD rats were randomly divided into control group (8 cases),AS group (10 cases) ,AS + SO2 group (10 cases).The rats in AS group and AS + SO2 group were given 700 000 U/kg Vitamin D3 and fed a high-cholesterol diet for 8 weeks to induce AS.Meanwhile, the rats in AS + SO2 group were intraperitoneally injected SO2 donor Na2SO3/NaHSO3 (0.54 mmol/kg,0.18 mmol/kg) every day.And the rats in control group and AS group were given the same dose of saline.After 8 weeks, the changes in atherosclerosis plaque size in the aortic root were observed by way of oil red O staining.Angiotensin Ⅱ (Ang Ⅱ) and endothelin-1 (ET-1) in the aortic homogenate were detected by using radioactive immunoassay.Results Compared with the control group, the atherosclerosis plaque size was markedly increased in AS group, while SO2 treatment significantly decreased the atherosclerosis plaque size in AS rats.Meanwhile,the content of Ang Ⅱ and ET-1 in the aortic homogenate from AS group were increased compared to those in the control group [(11.52 ±4.15) ng/g vs (5.46 ± 1.21) ng/g, (11.91 ± 4.93) ng/g vs (3.81 ± 1.21) ng/g,all P ＜0.01] ,while SO2 donor treatment markedly decreased the content of Ang Ⅱ and ET-1 in AS rats [(6.25 ± 2.85) ng/g, (8.35 ± 2.45) ng/g] (all P ＜ 0.01).Conclusions SO2 can play an important role in the regulation of vasoactive peptide Ang Ⅱ and ET-1 in AS rat aorta.This effect may be one of the mechanisms by which SO2 antagonize AS.