Effects Of Soluble Guanylate Cyclase Activator Cinaciguat In The Human Umbilical Vein Endothelial Cells Treated With High Glucose In Vitro

BackgroundVasculopathy because of its high incidence, morbidity and mortality becames one of the major complications of diabetes. Endothelial dysfunction is the key in the pathogenesis of vascular disease in diabetes mellitus.High glocose can damage endothelial function through inflammation,oxidative stress and so on. As a kind of signal transduction molecules, nitric oxide can play a role in vasodilator, anti-inflammatory,antioxidant through NO/sGC/cGMP signalling pathway. However, reactive oxygen species can oxidize NO and soluble guanylate cyclase(sGC) heme in pathological conditions,leading to the impaired NO/sGC/cGMP signalling pathway. Cinaciguat(CIN) is one of the new discovered NO-independent and haem-independent sGC activators. Several studies show that CIN has beneicial efects on myocardial infarction, myocardial ischemia/reperfusion injury, endothelial dysfunction which caused by oxidative stress,the formation of new blood vessels lining, myocardial hypertrophy.However,it is not clear whether CIN has an antioxidant and anti-inflammatory effect in the progress of diabetic impediments.Therefore, we intend to investigate the protective effect of cinaciguat in the human umbilical vein endothelial cells(HUVECs) treated with high glucose.ObjectiveTo observe the effects of cinaciguat on the high glucose-induced nitric oxide(NO), indicators of oxidative stress and inflammatory factor,explore that whether CIN can truly and effectively protect the injury of vascular endothelial cells and explored the possible underlying mechanisms,providing a new way of prevention for diabetic vascular complications.Methods1. The cells purchased were cultured in DMEM medium supplemented with 10% fetal bovine serum, in an atmosphere of 5% CO2 at 37℃.2. The damage model of HUVECs induced by high glucose in vitro:HUVECs were treated with high glucose (33.3mmol/L) for 48h to induce the injury.3. The effects of CIN on the high glucose-induced cell viability:After growing to 70%-80% confluence,HUVECs were treated with high glucose (33.3mmol/L) for 48h to induce the injury, then HUVECs were treated with CIN (0.01、0.1、1 和 10umol/L) for 48h. The cell viability was detected by MTT assay.4. The cultured HUVECs were treated with normal glucose (5.5mmol/L), normal glucose+CIN(1μmol/L),high glucose (33.3mmol/L), high glucose+CIN(1μmol/L). After 48h, the cells were collected for subsequent experiments.5.The NO is measured by nitrate reduction test.6. Production of ROS were measured using an oxidation sensitive fluorescent probe (DCFH-DA). Production of MDA,SOD activity was detected by TBA method and xanthine oxidase method.7. ICAM-1 and VCAM-1 mRNA expression in cells were measured by RT-PCR.8. VCAM-1 and ICAM-1,NF-κBp65 protein expression in cells were measured by Western blotting.Results1. In comparison with the normal glucose group, the cell viability of high glucose group(40.7±10.1%) was decreased(P＜0.05). Compared with high glucose group,the cell viability of CIN(0.01,0.1,1,10μmol/L) were significantly increased (60.0± 13.6)%,(79.1±11.6)%,(93.1±9.1)%,(93.4±9.3)%(all P<0.05). However,compared with CIN1μmol/L group,the cell viability of the CIN10μmol/L group was not significantly changed(P>0.05).2. Compared with the normal glucose group, there was significant decrease of NO from 18.26±2.03μmol/L to 12.78±0.92μmol/L in high glucose group(P<0.05).In comparison with high glucose group, the content of NO increased in high glucose+CIN group from 12.78±0.92μmol/L to 17.24±1.69μmol/L (P< 0.05).There was no change between the normal glucose group with normal glucose+CIN group(P>0.05).3. Compared with control group, in the high glucose group the production of ROS was increased from 1.00±0.00 to 2.57±0.44, the amount of MDA was increased from 1.32±0.37nmol/ml to 9.32±2.21nmol/ml, SOD activity was decreased from 22.82±4.17 U/ml to 10.79±2.66U/ml (P＜0.05).Whereas, pre-treatment with CIN decreased the production of ROS from 2.57±0.44 to 1.61±0.28, decreased the amount of MDA from 9.32±2.21nmol/ml to 4.22±1.55 nmol/ml, increased SOD activity from 10.79±2.66 U/ml to 18.09±3.36 U/ml (P＜0.05). There was no change between the normal glucose group with normal glucose+CIN group(P>0.05).4. Compared with the normal glucose group, there was significant increase expression of ICAM-1,VCAM-1 and NF-KBp65 in high glucose group(P<0.05).In comparison with high glucose group, the expression of ICAM-1,VCAM-1 and NF-κBp65 decreased in high glucose+CIN groups(P< 0.05). There was no change between the normal glucose group with normal glucose+CIN group(P>0.05).Conclusion1. High glucose accelerates the injury for the cultured human umbilical vein endothelial cell in vitro,cinaciguat can protect the injury of vascular endothelial cells.2. The cellular mechanisms of cells injury high glucose induced include decrease of NO, increase of oxidative stress and inflammatory factor.3. cinaciguat protects HUVECs from damage induced by hyperglycemia through increase of NO,reduce of ROS and MDA,elevation of SOD activity,inhibition of NF-κBp65,ICAM-1 and VCAM-1 expression.