| ObjectiveTo investigate the inhibitory effect of recombinant human angiotensin converting enzyme 2 on the proliferation and apoptosis of human renal mesangial cells induced by angiotensin II.MethodsHuman renal mesangial cells were cultured in low sugar Dulbecco’s modified eagle medium with 10% fetal serum bovine.Then they were randomly divided into 4 groups:control,Rh ACE2,Ang II,Ang II+ Rh ACE2.There is no Rh ACE2 or Ang II in the control group,while the Rh ACE2 group was supplied with Rh ACE2 at a concentration of 0.1 ng/μL and the Ang II group with Ang II at a concentration of 10-6mmol/L.And the medium of Ang II+Rh ACE2 group was supplemented with Ang II at a concentration of 10-6 mmol/L and 0.001,0.01 or 0.1 ng/μl Rh ACE2.The cell vitality was measured by MTT.While the cell cycle and cell apoptosis were determined by flow cytometry.Results(1)The cell viabilities of the control group,Rh ACE2 group and Ang II group were 0.90±0.08,0.88±0.01,1.27±0.09 respectively.And in Ang II+Rh ACE2 group,the cell viabilities were 1.15±0.10,1.04±0.10,0.91±0.01.Compared with the control group(0.90±0.08),the cell viability in Ang II group(1.27±0.09)was increased(P<0.05).Compared with Ang II group(1.27±0.09),the cell viabilities in Ang II+Rh ACE2 group(1.15±0.10,1.04±0.10,0.91±0.01)were decreased and there were significant differences when rh ACE2 at a oncentration of 0.01 or 0.1 ng/μl in Ang II+Rh ACE2 group(P<0.05).when Rh ACE2 at a concentration of 0.01 or 0.1 ng/μl in Ang II+Rh ACE2 group,the differences were significant(P<0.05).The differences between control and Rh ACE2 group was not evident.There were no significant differences between control group and in Rh ACE2 group about cell viabilities.(2)The rates of S phase of the control group,Rh ACE2 group and Ang II group were 38.22±2.77,36.9±0.58,46.57±1.69 respectively.And in Ang II+Rh ACE2 group,the rates of S phase were 43.37±0.63,43.10±2.74,40.49±1.66.Compared with the control group(38.22±2.77),the rate of S phase in Ang II group(46.57±1.69)was increased(P<0.05).Compared with Ang II group(46.57±1.69),the rate of S phase in Ang II+Rh ACE2 group(43.37±0.63,43.10±2.74,40.49±1.66)were decreased and there was a distinct difference between Ang II group and Ang II+Rh ACE2 group when supplied with Rh ACE2 at A concentration of 0.1 ng/μl(P<0.05).And the difference between control and Rh ACE2 group was not distinct on the rate of S phase.(3)The cell apoptosis percentage of the control group and Ang II group were 4.15±1.15,1.77±0.71 respectively.And in Ang II+Rh ACE2 group,the cell apoptosis percentage were 5.15±1.3,6.99±0.96,10.06±1.03.Compared with the control group(4.15±1.15),the cell apoptosis percentage in Ang II group(1.77±0.71)was decreased(P<0.05).The cell apoptosis percentage in Ang II+Rh ACE2 group(5.15±1.3,6.99±0.96,10.06±1.03)were increased compared with Ang II group(1.77±0.71).(P<0.05).ConclusionsAng II induced HRMCs proliferation.And Rh ACE2 inhibited Ang II-induced proliferation and contributed to the cell apoptosis.ObjectiveTo investigate the protective effects of recombinant human angiotensin converting enzyme 2 on human umbilical vein endothelial cells injury induced by Angiotensin II and to explore its possible mechanism.MethodsHuman renal mesangial cells were cultured in low sugar Dulbecco’s modified eagle medium with 10% fetal serum bovine.Then they were randomly divided into 4 groups: control,Rh ACE2,Ang II,Ang II+ Rh ACE2.There is no Rh ACE2 or Ang II in the control group,while the Rh ACE2 group with Rh ACE2 at a concentration of 0.1 ng/μL and the Ang II group with Ang II at a concentration of 10-6mmol/L.And the medium of Ang II+Rh ACE2 group was supplemented with Ang II at a concentration of 10-6 mmol/L and 0.001,0.01,or 0.1 ng/μL Rh ACE2.The cell vitality was measured by MTT.The cell cycle,apoptosis,and levels of ROS were measured by flow cytometry,.then IL-8,TNF-α,TGF-β1 were detected by Enzyme linked immunosorbent assay respectively.LDH was detected by a microplate reader.Results(1)The cell viabilities of the control group,Rh ACE2 group and Ang II group were 0.91±0.05,0.98±0.08,0.69±0.04 respectively.And the cell viabilities of Ang II+Rh ACE2 group were 0.73±0.01,0.79±0.06,0.92±0.06.Compared with the control group(0.91±0.05),the cell viabilities in Ang II group(0.69±0.04)was decreased(P<0.05).The cell viabilities in Ang II+Rh ACE2 group(0.73±0.01,0.79±0.06,0.92±0.06)were increased,compared with Ang II group(0.69±0.04),and there was a great difference when Rh ACE2 at a concentration of 0.1 ng/μl in Ang II+Rh ACE2 group(P<0.05).The difference between control and Rh ACE2 group was not distinct.(2)The rate of S phase of the control group,Rh ACE2 group and Ang II group were 26.7±3.89,29.75±3.20,19.71±0.70 respectively.And in Ang II+Rh ACE2 group,the rate of S phase were20.55±1.32,21.06±3.37,26.21±0.94.Compared with the control group(26.7±3.89),the rate of S phase in Ang II group(19.71±0.70)was increased(P<0.05).Compared with Ang II group(19.71±0.70),the rate of S phase in Ang II+Rh ACE2 group(20.55±1.32,21.06±3.37,26.21±0.94)were decreased and when Rh ACE2 at a concentration of 0.1 ng/μl in Ang II+Rh ACE2 group,the differences were significant(P<0.05).There were no distinct difference between control group and Rh ACE2 group on the rate of S phase.(3)The cell apoptosis percentage of the control group,Rh ACE2 group and Ang II group were 2.32±0.31,1.88±0.03,11.01±0.18 respectively.And in Ang II+Rh ACE2 group,the cell apoptosis percentage were 8.64±0.37,7.24±0.33,5.84±0.15.Compared with the control group(2.32±0.31),the cell apoptosis in Ang II group(11.01±0.18)was increased(P<0.05).Compared with Ang II group(11.01±0.18),the cell apoptosis percentage in Ang II+Rh ACE2 group(8.64± 0.37,7.24±0.33,5.84±0.15)were decreased(P<0.05).There were no significant difference between control group and Rh ACE2 group(1.88±0.03)on cell apoptosis percentage.(4)The intracellular reactive oxygen species(ROS)was detected by flow cytometry.The ROS percentage of the control group,Rh ACE2 group and Ang II group were 14.89±1.33,13.45±0.88,90.46±2.20 respectively.And in Ang II+Rh ACE2 group,the percentage were 89.74±0.80,80.27±2.75,71.02±0.69.The percentage in Ang II group(90.46±2.20)was increased compared with the control group(14.89±1.33)(P<0.05)..Compared with Ang II group(90.46±2.20),the percentage in Ang II+Rh ACE2 group(89.74±0.80,80.27±2.75,71.02±0.69)were decreased and there was great significant difference between Ang II group and Ang II+Rh ACE2 group with Rh ACE2 at a concentration of 0.01 or 0.1 ng/μl P<0.05).There were no evident difference between control group and Rh ACE2 group(13.45±0.88)on The intracellular reactive oxygen species percentage.(5)The quantity of IL-8 in the cell culture supernatant of the control group,Rh ACE2 group and Ang II group were46±3.6,46.13±1.97,184.13±2.27 pg/ml respectively.The content in Ang II+Rh ACE2 group were 183.47±3.00,142.8±2.12,98.93±0.83 severally.Compared with the control group(46±3.6),the quantity in Ang II group(184.13±2.27)was increased(P<0.05).Compared with Ang II group(184.13±2.27),the quantity in Ang II+Rh ACE2 group(183.47±3.00,142.8±2.12,98.93±0.83)were decreased and when Rh ACE2 at a concentration of 0.001 or 0.1 ng/μl in Ang II+Rh ACE2 group,the differences were significant(P<0.05).There were no significant differences between control group and in Rh ACE2 group(46.13±1.97)on the quantity of IL-8 in the cell culture supernatant.(6)The quantity of TNF-α in the cell culture supernatant of the control group,Rh ACE2 group and Ang II group were49.45±0.66,48.44±0.44,153.50±0.25 pg/ml respectively.And in Ang II+Rh ACE2 group,the quantity were 152.63±1.33,145.68±0.25,102.34±0.44.Compared with the control group(49.45±0.66),the quantity in Ang II group(184.13±2.27)was increased(P<0.05).Compared with Ang II group(153.50±0.25),the quantity in Ang II+Rh ACE2 group(152.63±1.33,145.68±0.25,102.34±0.44)were decreased and when Rh ACE2 at a concentration of 0.001 or 0.1 ng/μl in Ang II+Rh ACE2 group,the differences were significant(P<0.05).There were no significant differences between control group and in Rh ACE2 group(48.44±0.44)on the quantity of TNF-α in the cell culture supernatant.(7)The quantity of TGF-β1 in the cell culture supernatant of the control group,Rh ACE2 group and Ang II group were27.38±2.43,30.18±1.21,97.41±1.21 pg/ml respectively.And in Ang II+Rh ACE2 group,the quantity were 93.21±6.42,88.31±5.29,46.29±5.29.Compared with the control group(27.38±2.43),the quantity in Ang II group(97.41±1.21)was increased(P<0.05).Compared with Ang II group(97.41±1.21),the quantity in Ang II+Rh ACE2 group(93.21±6.42,88.31±5.29,46.29±5.29)were decreased and when Rh ACE2 at a concentration of 0.1 ng/μl in Ang II+Rh ACE2 group,the differences were significant(P<0.05).There were no significant differences between control group and in Rh ACE2 group(30.18±1.21)on The the quantity of TGF-β1 in the cell culture supernatant.(8)The quantity of LDH in the cell culture supernatant of the control group,Rh ACE2 group and Ang II group were 227.91±7.99,214.07±7.99,490.89±15.98U/L respectively.And in Ang II+Rh ACE2 group,the quantity were 477.04±15.98,444.75±21.21,292.50±7.99.Compared with the control group(227.91±7.99),the quantity in Ang II group490.89±15.98)was increased(P<0.05).Compared with Ang II group(490.89±15.98),the quantity in Ang II+Rh ACE2 group(477.04±15.98,444.75±21.21,292.50±7.99)were decreased and when Rh ACE2 at a concentration of 0.001 or 0.1 ng/μl in Ang II+Rh ACE2 group,the differences were significant(P<0.05).There were no significant differences between control group and in Rh ACE2 group(214.07±7.99)on the quantity of LDH in the cell culture supernatant.ConclusionsAng II induces the apoptosis of HUVECs and release of inflammatory mediator.Rh ACE2 can inhibit the detrimental effects of Ang II. |
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