Protective Effect Of Isoorientin On DNA Damage And Cell Cycle Arrest In Rat Renal Tubular Epithelial Cells

Cadmium(Cd)is a kind of environmental heavy metal pollutant that seriously endangers public health and safety.It is widely present in the environment and is not easy to degrade,which can damage or even cancer multiple tissues.The kidney is an important metabolic organ of the body,which is extremely sensitive to cadmium.Different doses and time will cause different degrees of damage to the kidney.Isoorientin(ISO)is a natural plant extract component that has functions of antioxidant,antiviral.DNA oxidative damage is a process in which various stimuli cause oxidative responses to cells and induce DNA damage.Cell cycle arrest is the process by which cells respond to DNA damage.Based on the establishment of an in vitro model,this study studied the oxidative damage and cell cycle of cadmium-induced renal tubular epithelial cells,and explored the protective effect of isoorientin.1.Effect of cadmium on DNA oxidative damage in rat renal tubular epithelial cellsIn this study,rat renal tubular cell line NRK-52E and rat renal tubular primary cell rPT cells were selected as subjects.After treatment with different concentrations of cadmium(0,1.25,2.5,5 μmol/L):(1)nuclear morphological changes were observed with transmission electron microscopy(TEM)and nuclear DNA damage was detected by single cell gel electrophoresis assay;(2)expression of p-H2AX was detected by western blot and immunofluorescence;(3)changes of ROS and 8-OHdG level were measured with flow cytometry(FCM)and ELISA assay,respectively.The results showed that:compared with the control group(1)the nucleus damage and chromosome breakage of the cadmium group showed obvious damage,deformation damage of the nucleus;(2)the expression of p-H2AX was significantly increased(P<0.01),and there was obvious comet-like tailing phenomenon;(3)The levels of ROS and 8-OHdG were significantly increased(P<0.01)and were concentration dependent.It indicates that cadmium can cause DNA damage in NRK-52E and rPT cells,and this damage is related to oxidative stress.2.Effect of cadmium on cell cycle distribution of rat renal tubular epithelial cellsThe following experiments were carried out with NRK-52E and rPT cells as follows:(1)after treatment with different concentrations of cadmium(0,1.25,2.5,5 μmol/L)for 12 h,the flow cytometry was used to detect the cell cycle;(2)after treatment,the expression of p53,p21 and cell cycle-associated regulatory proteins was detected by Western-blot;(3)after adding 20μmol/L p53 inhibitor(Pifithrin-α,PFT-α)and 2.5 μmol/L cadmium alone or in combination,western-blot was used to detect the expression of p53,p21 and related cyclins.The results showed that:(1)compared with the control group,the number of cells in the cadmium treatment group increased significantly in the G1 phase(P<0.01),and the concentration gradient was dependent on a certain range;(2)compared with the control group,The expression of p53 and p21 protein in 2.5,5 μmol/L cadmium group was significantly increased(P<0.01),the expression of Cyclin D1,CDK4 and Cyclin E1,CDK2 was significantly decreased(P<0.01);(3)compared with the PFT-a and cadmium co-treatment group,the expression levels of p53 and p21 protein were significantly decreased(P<0.01),and the expression levels of Cyclin D1,CDK4 and Cyclin E1 and CDK2 were significantly increased(P<0.01).The results indicate that cadmium can induce G1 phase arrest by p53-dependent pathway.3.Protective effect of Isoorientin on cadmium-induced injury of rat renal tubular epithelial cellsNRK-52E cells were used as in vitro model to perform present study.The effect of different concentration of isoorientin on cell viability were screened by CCK-8 assay and RTCA system,and 80 μmol/L isoorientin was used in the following studies.NRK-52E cells treated with 2.5μmol/L cadmium(Cd)in presence or absence with 80 μM isoflurane for 12 h,and performed the following experiments:(1)nuclear morphological changes were observed with transmission electron microscopy(TEM)and nuclear DNA damage was detected by single cell gel electrophoresis assay;(2)changes of ROS and 8-OHdG level were measured with flow cytometry(FCM)and ELISA assay,respectively;(3)expression of p-H2AX was detected by western blot and immunofluorescence;(4)cell cycle distribution and cycle-associated proteins expression were checked by FCM and western blot,respectively.The results showed that,isoorientin and Cd co-treatment group compared to Cd treatment group,(1)the nucleus damage and chromosome breakage were alleviated which presented as more regular nuclear morphology and shorter comet-tail length,respectively;(2)the level of ROS and 8-OHdG were significantly decreased(P<0.01);(3)p-H2AX protein level was significantly decreased(P<0.01)presented by densitometry analysis and fluorescence intensity;(4)the number of cells in the G0/G1 phase was significantly decreased(P<0.01),the expression of p53 and p21 were significantly decreased(P<0.01)while Cyclin D1,CDK4,Cyclin E1 and CDK2 proteins expression level were increased(P<0.05).These data illustrate that isoorientin alleviates DNA oxidative damage and cycle arrest induced by Cd in renal tubular epithelial cells.Conclusions,cadmium can induce DNA damage in rat renal tubular epithelial cells by inducing ROS production,and cell cycle arrest in G1 phase through p53-dependent pathway.Isoorientin can alleviate cadmium-induced DNA oxidative damage and cell cycle arrest in rat renal tubular epithelial cells through its antioxidant activity.