| Part Ⅰ: Changes of mitochondrial function、PGC-1α and NFAT in different degree of inflammatory vascular endothelial cell injuryObjective: To observe the changes of mitochondrial function 、 peroxisome proliferator-activated receptor-γ coactivator-1α(PGC-1α)and activated T cell nuclear factor in TNF-α-induced injury of vascular endothelial cells.Methods: 1,Different concentrations of tumor necrosis factor(TNF-α)(10,20,40,80 ng/ml)to stimulate human umbilical vein endothelial cells(HUVECs)12,24,36 hours to prepare different degrees of vascular endothelial cell injury model,CCK-8 method was used to detect the cell viability.Apoptosis was detected by Annexin V-FITC/PI double staining method.Morphological changes were observed under inverted optical microscope to determine whether the model was established successfully.2,The mitochondrial membrane potential was detected by JC-1 immunofluorescence staining and fluorescence microscopy.Mito SOX? Red staining combined with fluorescence microscopy was used to detect mitochondrial reactive oxygen species.3,RT-q PCR,Western blot method to detect m RNA and protein levels of PGC-1α,activated T cell nuclear factor 1(NFAT1),activated T cell nuclear factor 2(NFAT2).Results: 1,Different degrees of vascular endothelial cell injury model was successfully established.CCK-8 results showed that compared with normal cells,with the increase of TNF-α concentration and the role of time,HUVECs cell activity decreased gradually,and at the concentration of 40 ng/ml,the stimulation time was 24 hours,decreased by about 49%.The difference was statistically significant(P <0.05).Since the concentration of TNF-α was 10ng/ml,the cell viability did not change significantly compared with the normal cell group.TNF-α stimulated concentration was changed to 20,40,80 ng/ml,stimulation time of 24 hours.After 20,40,80 ng/ml TNF-α stimulated HUVECs for 24 h,The apoptotic rate of HUVECs increased gradually,and the difference was statistically significant(P <0.05).The morphological changes were observed under light microscope.The results showed that the cells in the control group were normal,the morphology was polygonal,spindle shape and pavement.20 ng/ml TNF-α treatment group,the morphological changes were not obvious.When the concentration of TNF-α was 40 ng/ml,the cell morphology was elongated and the cell gap increased,When the TNF-α concentration is 80ng/ml,cell morphological changes more obvious,and even some cells can be seen pseudo-foot extension.2,Mitochondrial function changes: Jc-1 immunofluorescence staining with mitochondrial membrane potential was measured by fluorescence microscopy.Mito SOX ? Red staining combined with fluorescence microscopy was used to detect mitochondrial reactive oxygen species,The results showed that compared with the control group(JC-1: 2.74 ± 0.06;mt ROS: 1.86 ± 0.12),with the increase of TNF-α stimulation concentrationthe,the mitochondrial membrane potential decreased gradually(1.77 ± 0.08,0.51 ± 0.09,0.23 ± 0.12),and the mt ROS gradually increased(2.97 ± 0.14,3.36 ± 0.14,4.28 ± 0.19),the difference were statistically significant(P <0.05).3,RT-q PCR and Western blot were used to detect the expression of PGC-1α m RNA and protein in HUVECs after TNF-α(20,40,80 ng/ml)treated for 24 hours.The results showed that compared with the control group(m RNA: 1.00 ± 0.09;protein: 2.16 ± 0.05),the level of PGC-1α m RNA and protein in each experimental group decreased gradually(m RNA: 0.51 ± 0.05,0.08 ± 0.04,0.02 ± 0.01;protein: 1.56 ± 0.05,0.78 ± 0.08,0.50 ± 0.12),the difference was statistically significant(P <0.05).4,RT-q PCR and Western blot were used to detect the expression of NFAT1、NFAT2 m RNA and protein in HUVECs after 24 hours of TNF-α(20,40,80 ng/ml)treated.The results showed that compared with the control group(NFAT1: m RNA 1.00 ± 0.06,protein 0.54 ± 0.12;NFAT2: m RNA 1.00 ± 0.08,protein 0.09 ± 0.02),the m RNA and protein levels of NFAT1 and NFAT2 were significantly increased in each experimental group(NFAT1: m RNA 0.91 ± 0.07,1.97 ± 0.16,2.93 ± 0.01,protein 0.50 ± 0.04,0.95 ± 0.46,1.18 ± 0.09;NFAT2: m RNA 1.96 ± 0.09,2.85 ± 0.43,3.86 ± 0.15,protein 0.17 ± 0.03,0.36 ± 0.13,0.64 ± 0.06),When the stimulation concentration were 40 ng/ml and 80 ng/ml,the difference was statistically significant(P <0.05).Conclusion:1,Different concentrations of TNF-α(20,40,80 ng/ml)can cause different degrees of HUVECs cell damage.2,Mitochondrial dysfunction,NFAT expression may be involved in TNF-α-induced HUVECs cell damage mechanism.Part Ⅱ: The effects of PGC-1α on TNF-α-induced vascular endothelial cell injury and NFAT expression and its mechanismObjective: To investigate the effect of PGC-1α on TNF-α-induced vascular endothelial cell injury and NFAT expression,and to explore its mechanism.Methods: HUVECs were cultured in vitro,HUVECs cells were transfected with Lentivirus-si-PGC-1α or Lentivirus-PGC-1α to knock down and overexpress PGC-1α,respectively.Experiment were divided into si-Null group,si-PGC-1α group,Lv-Null group and Lv-PGC-1α group.In addition,HUVECs coming from Lv-Null group and Lv-PGC-1α group were treated with TNF-α at the concentration of 40 ng/ml.We detected cell apoptosis by Annexin V-FITC/PI double staining method and cell viability by CCK-8 method,Morphological changes were observed under inverted optical microscope.Mitochondrial ROS was analyzed with Mito SOX? Red Kit,and intracellular calcium levels with Fura 3-AM kit,RT-q PCR and Western blot were analyzed to evaluate the m RNA and protein expression levels of PGC-1α,NFAT1,NFAT2.Results:1,Transfection efficiency is up to 90% or more when Lentivirus MOI was 20.2,(1)PGC-1α gene silencing group: Compared with the control group,PGC-1α gene silencing group HUVECs cell activity decreased(0.71±0.84vs1.00±0.04),and the apoptosis increased(7.43%±0.83%vs4.63%±0.04%),all P <0.05.Cell gap was lower than the control group decreased,pseudopodia increased.Intracellular mitochondrial ROS levels increased(1.61±0.09vs0.81±0.14),intracellular Ca2+ concentration increased(4.27±0.27vs2.73±0.19),NFAT1,NFAT2 m RNA and protein expression levels were increased(NFAT1:m RNA 2.43±0.02 vs1.00±0.02,protein 0.95±0.01 vs0.68±0.02;NFAT2:m RNA 2.16±0.05 vs1.00±0.05,protein 0.91±0.02 vs0.73±0.03),all P <0.05.(2)PGC-1α overexpression group: After 40ng/ml TNF-α treatment,the cell activity of PGC-1α overexpression group was higher than that of the control group(0.80±0.13vs0.59±0.01),the cell apoptosis was decreased(12.1%±0.04%vs21.3%±0.07%)and the cell morphology changed slightly.In PGC-1α overexpression group,mitochondrial ROS levels decreased(3.29±0.47vs5.90±0.76)and intracellular Ca2+ concentration decreased(5.24±0.24vs6.31±0.26),NFAT1,NFAT2 m RNA and protein expression levels were reduced(NFAT1:m RNA 0.72±0.02 vs1.00±0.01,protein 0.72±0.03 vs0.95±0.02;NFAT2:m RNA 0.67±0.01 vs1.00±0.01,protein 0.76±0.02 vs0.95±0.03),all P <0.05.Conclusion:1,PGC-1α has protective effect on TNF-α-induced injury of HUVECs cells.2,PGC-1α can reduce the level of mitochondrial ROS in TNF-α-induced HUVECs.3,PGC-1α may reduce the intracellular mitochondrial ROS levels,Thereby inhibiting Ca2+/NFAT pathway to protect TNF-α-induced HUVECs cell damage. |
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