N-Acetyl-cysteine Protects Endothelial Nitric Oxide Synthase Activity In Human Umbilical Vein Endothelial Cells And Its Molecular Mechanism

Objective: To observe effects of N-Acetyl-cysteine(NAC)preconditioning on endothelial nitric oxide synthase(eNOS)activity,therefore to explore the mechanism of NAC preconditioning protects the eNOS activity in human umbilical vein endothelial cells(HUVECs)induced by angiotensin?(Ang?)and Palmitic acid(PA)stimulation.Methods: HUVECs were cultured with Ang?(1×10-7M,1×10-6M)for 12 h and PA(25?M,50?M,100?M,200?M for 24 h and 100?M for 12 h,24h,36h)after the cells were identified by SABC method.In addition,cells were pretreated with ROS scavenger(NAC),HUVECs were randomly divided into normal control group,Ang? group,NAC pretreatment group,NAC pretreatment+Ang? group(1×10-3M,NAC pretreated the cells 1h before Ang?stimulation)and vehicle control group,PA(50?M,100?M for 24h)group,NAC pretreatment group,NAC pretreatment+PA group(1×10-3M,NAC pretreated the cells 1h before PA stimulation).The levels of eNOS,PP2A-C?,I2PP2 A protein expression and p-eNOS Ser1177,p-PP2A-c Y307 were measured by Western Blot.The activity of constitutive NOS(cNOS)and the content of NO in cell culture medium were determined via chemical colorimetric methods.Results:(1)Compared with control group,the levels of eNOS and PP2A-C? protein expression in Ang? group were unchanged in each group,the level of I2PP2 A protein expression,p-eNOS Ser1177 and p-PP2A-c Y307 level decreased significantly(p<0.05),the activity of cNOS and the content of NO in cell culture medium decreased significantly(p<0.05);Compared with vehicle control group,the levels of eNOS,PP2A-C?,I2PP2 A protein expression and p-PP2A-c Y307 level in PA group were unchanged in each group,p-eNOS Ser1177 level decreased significantly(p<0.05),the activity of cNOS and the contentof NO in cell culture medium decreased significantly(p<0.05).(2)Compared with same concentration Ang? group,NAC pretreatment significantly upregulated the level of I2PP2 A protein expression(p<0.05),p-eNOS Ser1177 and p-PP2A-c Y307 levels increased(p<0.05),meanwhile the activity of cNOS and the content of NO in cell culture medium increased(p<0.05),but the levels of eNOS and PP2A-C? protein expression were unchanged in each group;Compared with same concentration PA group,NAC pretreatment significantly upregulated p-eNOS Ser1177(p<0.05),meanwhile the activity of cNOS and the content of NO in cell culture medium increased(p<0.05),but the levels of eNOS,PP2A-C?,I2PP2 A protein expression and p-PP2A-c Y307 were unchanged in each group.Conclusion:(1)NAC pretreatment can protect the activity of eNOS induced by Ang?/PA stimulation,and increase the production of NO,thus play a protective role in endothelial cells,but the molecular mechanisms are different.(2)Both Ang? and PA can decrease the activity of eNOS in HUVECs,decrease NO production,then impair endothelial function.But the molecular mechanisms are different,Ang? may increase the activity of PP2 A through upregulating of PP2A-c Y307 and I2PP2 A,resulting in the decrease of eNOS activity,PA may lead to the decrease of eNOS activity through other mechanisms.