Protective Effects Of 4-ISO-Tempol On Rat Glomerular Mesangial HBZY-1 Cells Against Oxidative Stress Under High Glucose And Its Mechanisms

Objective:4-isothiocyanate-2,2,6,6-tetramethyl piperidinooxyl(4-ISO-Tempol)is one kind of piperidine nitroxide compounds with strong antioxidant activity.It has great potential for clinical application in the protection of ionizing radiation and DNA damage,the prevention and treatment of cancer,hypertension,weight control,cardiomyopathy and so on.In this paper,we mainly discussed the effects of 4-ISO-Tempol on rat glomerular Mesangial HBZY-1 cell proliferation under high glucose conditions and its related mechanisms,and also provided certain reference value for clinical further research.Methods: The sulforhodamine B(SRB)was used to detect the effects of 4-ISO-Tempol on the proliferation viability of HBZY-1 cells.The SOD,CAT,GSH-Px,MDA assay kits were used to determine the intracellular SOD,CAT and GSH-Px enzyme activities,and MDA level in HBZY-1 cells.Cell apoptosis and cycle of HBZY-1 cells were analysed by flow cytometry.The intracellular ROS generation in HBZY-1 cells was detected by fluorescence probe assay,and the protein expressions of ERK1/2,p-ERK1/2,P38,p-P38,IKK,p-IKK,and Ncedherin in HBZY-1 cells were done using western blotting assay.Results: The results showed that 4-ISO-Tempol(concentration of 1.25,2.5,5,10,20 μg/mL)had significant inhibitory effects on the proliferation viability of HBZY-1 cells cultured under normal conditions.The inhibition rates of 24 h,48 h and 72 h were 2.20%,8.50%,26.65%,61.23%,82.03%;3.50%,7.13%,28.87%,56.16%,80.39%;and 4.07%,9.69%,31.06%,65.89%,87.91%,respectively.The growth rate of HBZY-1 cells was significantly increased under the condition of 30 M high glucose,while the 4-ISO-Tempo(0.313,0.625,1.25,2.5 mu g/mL),which had no obvious effect on the proliferation of HBZY-1 cells under normal conditions,obviously inhibited the growth of high glucose induced HBZY-1 cells.4-ISO-Tempol had no effects on the apoptosis and cycle of HBZY-1 cells under high glucose conditions.Moreover,4-ISO-Tempol significantly increased the activities of SOD,CAT and GSH-Px in the HBZY-1 cells under high glucose conditions,and reduced the production of MDA content and ROS level.It also inhibited the protein expressions of p-P38,IKK,p-IKK and Ncadherin in HBZY-1 cells and had no effect on P-ERK1/2 expression under high glucose condition.Conclusion: 4-ISO-Tempol can obviously inhibit HBZY-1 cells proliferation induced by high glucose,and its mechanism may be related to the enhancement of intracellular anti-oxidant stress and the inhibition of the NF-κB/IKK signaling pathway.