| Objective To investigate the effect of angiotensin(1-7)(Ang1-7)and its possible mechanism by cultured human glomerular endothelial cells(HGECs)in vitro,Screening the appropriate concentration and time of angiotensin ?(Ang ?)on HGECs.METHODS 1.Cultured human glomerular endothelial cells: the cells were cultured with DMEM(Low Glucose)medium containing 15% fetal bovine serum and cultured in a incubator(saturated humidity,5% CO2,37 ?).The culture medium replaced with every 24 hours.The cells was digested and passed with 0.25 % trypsin –EDTA,After the cells reached about70%-80%.2.Cultured HGECs were randomly divided into 6 groups according to different intervention factors: control group(without intervention factors),Ang ? group,Ang1-7 group,Ang ? +Ang1-7 group(the Ang1-7 was pretreated for 30 min before Ang ?was added in).Ang ? +Ang1-7+Ang1-7 Mas receptor blocker group(A779 was pretreated with 15 min first,then adding Ang1-7 to interfere with 30 min,and then added to Ang ? treatment),the Mas receptor blocker group(A779 group).all groups were replaced with serum-free medium culture for 12 hours before experiment.3.The cells viability and Cytotoxicity were measured by CCK-8,then screened the appropriate concentration and time of Ang ?,Ang1-7,A779.4.The apoptotic rate of HGECs were detected with flow cytometry.The cells were labeled with DCFH-DA detect the content of reactive oxygen species(ROS)by flow meter.The changes of ROS in the cells were shoted by fluorescence microscopy.5.The content of lactate dehydrogenate dehydrogenase(LDH)in HGECs culture medium was determined by microplate method,and the level of nitric oxide(NO)in supernatant was determined with nitrate reductase method to reflect indirectly.The levels of Endothelin 1(ET-1),Interleukin-6(IL-6),Tumor necrosis factor alpha(TNF-?),Transforming growth factor beta1(TGF-b1),monocyte chemoattractant protein-1(MCP-1),intercellular adhesion molecule-1(ICAM-1)in the supernatant of cell culture were measured with Enzyme-linked immuno sorbent assay(ELISA).RESULTS 1.Compared with the control group,the significant difference of HGECs viability through the CCK-8 experiment was taken as the optimal concentration and time of Ang ?(1umol/L,24h).In the presence of Ang ?,Ang1-7was used to determine the cells activity,Also A779 determine in the presence of Ang ?+Ang1-7,and the group with significantly different concentrations was the best.The Ang1-7 was selected 0.1 umol/L,A779 was 10 umol/L.2.Compared with control group,the apoptotic rate and the average fluorescence intensity of ROS,IL-6,TNF-a,TGF-b,MCP-1,sICAM-1were increased in Ang ?(1 mmol/L)group(P<0.05).Compared with Ang ?(1 mmol/L)group,the apoptotic rate and ROS,IL-6,TNF-a,TGF-b,MCP-1,s ICAM-1 levels in Ang ?+Ang1-7(1 +0.1 mmol/L)group were decreased significantly(P < 0.05).Compared with Ang ?+ Ang1-7(1 +0.1 mmol/L)group,the addition of A779(10 mmol/L)increased the cell apoptosis rate,the production of ROS and the content of IL-6,TNF-?,TGF-?1,MCP-1,s ICAM-1 in the supernatant of cell culture(P < 0.05).3.In the presence of Ang ?(1 mmol/L)group,the addition of Ang1-7(10,1,0.1,0.01 ?mol/L)inhibited the release of ET-1,LDH and promoted the release of NO in a dose-dependent manner(P < 0.05).CONCLUSION 1.Ang ? could increase the apoptosis rate of HGECs,the production of ROS,and increase the expression of inflammatory factors.2.Ang1-7 could decrease the apoptosis rate of HGECs,the production of ROS,and the expression of inflammatory factors induced by Ang ? through Mas receptor.3.In the presence of Ang ?,the addition of Ang1-7 inhibited the HGECs damage induced by Ang ? in a dose-dependent manner,in which the leakage of LDH,the secretion of ET-1 decreased and the release of NO.The protective effect of Ang1-7 was almost completely eliminated by antagonist A779. |
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