The Role And Mechanism Of Parthanatos In Cadmium-induced Oxidative Damage And Apoptosis Of Rat Renal Tubular Epithelial Cells

Cadmium is a kind of heavy metal pollutant with strong accumulation,and it widely exists in industrial as well as living environment,which has a serious impact on human and animal health.Poly-ADP-ribose polymerase-1(PARP-1)is a major member of the poly-ADP-ribose polymerase family,which has the function of specifically identifying and repairing DNA damage,however,if PARP-1 is overactivated,it will also induce Parthanatos in cells.Parthanatos is a way of cell death based on PARP-1 activation,its main characteristics are the excessive activation of PARP-1 leading to the accumulation of poly-ADP ribose(PAR)in the cytoplasm,changes in mitochondrial permeability and nuclear translocation of apoptosis-inducing factors(AIF),DNA fragmentation and rapid decrease in intracellular NAD+ as well as ATP levels.At present,the research on Parthanatos is still in the early stage,although it has been found that involved in Parkinson’s disease,diabetes,heart failure and other diseases,the specific molecular mechanism of Parthanatos is not clear,and no studies have shown whether Parthanatos is related to the toxicological mechanism of heavy metals.In recent years,the study of our research group shown that cadmium can activate PARP protein,ERK1/2 MAPK,and induce oxidative stress and apoptosis in a variety of cells,but the role of PARP-1 in this process and the relationship between ERK1/2 and Parthanatos remain unclear.Therefore,in this study,rat renal tubular epithelial cell line(NRK-52E cells)and primary rat renal tubular epithelial cell(rPT cells)were used as cell model and combined with in vivo experiment,to explore whether PARP-1-dependent Parthanatos is involved in cadmium-induced oxidative damage and apoptosis of rat renal cells,and the mechanism of Parthanatos as well as the role of ERK1/2 were further revealed.The research content is divided into the following parts:1.Cadmium-induced oxidative damage and apoptosis in rat renal tubular epithelial cellsIn order to study whether cadmium can induce oxidative stress and apoptosis in rat renal tubular epithelial cells,NRK-52E and rPT cells were used as research models.The effects of different concentrations of cadmium on the proliferation of NRK-52E cells were detected by RTCA and cell scratch test,besides,the effect of cadmium on survival rate of rPT cells was detected by CCK-8.Observe the morphological changes of the cells with a scanning electron microscope.The expression of DNA damage and apoptosis related proteins were detected by qRT-PCR,Western Blot and immunofluorescence.At the same time,oxidation index related test kits were used to detect the changes of cell oxidation level,the flow cytometry was used to detect the content of reactive oxygen species,cycle distribution and apoptosis.The results showed that the proliferation activity of cells was inhibited,the cell morphology and DNA were damaged after exposure to cadmium.In addition,with the increase of cadmium concentration,the contents of reactive oxygen species and malondialdehyde increased significantly,while the activities of related antioxidant enzymes decreased significantly(P<0.05 or P<0.01).Besides,the expression of cell cycle-related proteins such as Cyclin D1,Cyclin E1,CDK4 and CDK2 decreased significantly(P<0.05 or P<0.01),and the apoptosis rate increased in a dose-dependent manner.These results illustrated that cadmium exposure can cause oxidative damage and apoptosis.2.Cadmium induces PARP-1-dependent Parthanatos in rat renal tubular epithelial cellsTo explore whether cadmium exposure can induce PARP-1-dependent Parthanatos in rat renal tubular epithelial cells,Western Blot and immunofluorescence were used to detect the expression or nuclear translocation of PARP-1 as well as Parthanatos-related proteins,the changes of ATP and NAD+content were detected by related kits,and the changes of Parthanatos related indexes were detected after the transcription level of PARP-1 gene was knocked down by SiRNA.The results showed that with the increase of cadmium concentration and incubation time,the expression of PARP-1 and AIF increased significantly,while the content of ATP and NAD-decreased significantly(P<0.05 or P<0.01).After inhibiting the expression of PARP-1,the expression of PAR and AIF decreased significantly,and the nuclear translocation of AIF was also inhibited significantly(P<0.01).These results illustrated that PARP-1-dependent Parthanatos was also activated in the process of oxidative damage and apoptosis of rat renal tubular epithelial cells induced by cadmium.3.The role of Parthanatos in oxidative damage and apoptosis of rat renal tubular epithelial cells induced by cadmiumIn order to investigate the role of Parthanatos in oxidative damage and apoptosis of rat renal tubular epithelial cells induced by cadmium,Parthanatos-dependent protein PARP-1 siRNA and specific inhibitor DPQ were used to inhibit the expression of PARP-1,and then NRK-52E cells were treated with low concentration(2.5 μM)or high concentration(10 μM)cadmium for 12 h.The changes of oxidative damage,the expression of cell cycle and apoptosis-related proteins were detected by immunofluorescence or Western Blot.Besides,the changes of cell cycle distribution,mitochondrial membrane potential,the level of reactive oxygen species and apoptosis were detected by flow cytometry.The results showed that damage of DNA and cytoskeleton induced by 10 μM cadmium was significantly higher than that caused by 2.5 μM cadmium,inhibition of PARP-1 expression in 2.5 μM cadmium treatment would aggravate DNA damage,while it would reduce DNA damage in 10 μM cadmium treatment.After cadmium treatment,the mitochondrial membrane potential decreased significantly,and the content of ROS increased significantly(P<0.05 or P<0.01).Compared with the cadmium treated group,the mitochondrial membrane potential increased significantly,and the content of ROS decreased significantly after intervention of PARP-1siRNA and DPQ(P<0.01).In addition,cell cycle arrest was induced by cadmium exposure for 12 h,in which 2.5 μM induced G0/G1 phase arrest,while 10 μM induced S phase arrest.After inhibiting the expression of PARP-1,the degree of cell cycle arrest was aggravated in 2.5 μM cadmium treatment group and alleviated in 10 μM cadmium treatment group.Furthermore,inhibiting the activation of PARP-1,the nuclear translocation of AIF and Cyt C inhibited significantly that induced by cadmium,the expression of Cleaved caspase-9/3 and apoptosis rate decreased significantly.These results illustrated that when cadmium exposure caused low DNA damage,PARP-1 could repair DNA damage and inhibit cell cycle arrest and apoptosis,while cadmium caused severe DNA damage,PARP-1 would induce Parthanatos and aggravate cell cycle arrest,resulting in apoptosis.In addition,the activation of Parthanatos will aggravate the oxidative damage induced by cadmium,and the inhibition of PARP-1 overexpression will contribute to cell survival.4.The role of ERK1/2 in Parthanatos and apoptosis of rat renal tubular epithelial cells induced by cadmiumIn order to investigate the effects of ERK1/2 on Parthanatos and apoptosis during cadmium-treated rat renal tubular epithelial cells,the effect of cadmium on the activation of ERK1/2 was detected by Western Blot,immunofluorescence and flow cytometry.Then the specific inhibitor U0126 was used to inhibit the activation of ERK1/2 induced by cadmium and the changes of related indexes were detected,so as to explore the effect of ERK1/2 on Parthanatos and apoptosis and its mechanism.The results showed that cadmium exposure significantly upregulated the expression of PARP-1 and AIF by activating ERK1/2(P<0.01),in addition,cadmium exposure not only induced apoptosis of rat renal tubular epithelial cells,but also promoted autophagy,while ERK1/2 could inhibit cell apoptosis by up-regulating the level of autophagy.These results illustrated that ERX1/2 can induce Parthanatos by up-regulating the level of PARP-1 as well as inhibit apoptosis by promoting the level of autophagy.5.Cadmium-induced renal oxidative damage and apoptosis in SD rats and the effect of PARP-1To further verify the oxidative damage of cadmium on rat kidney cells and the expression of PARP-1,α-lipoic acid was selected as antioxidant to carry out the related test Details as follows:24 female SD rats of 60-70g were randomly divided into four groups after pre-feeding for 1-week,normal control group,cadmium group,α-lipoic acid+cadmium group and α-lipoic acid control group.Rats in the control group were free to drink DIW,rats exposed to cadmium were free to drink the prepared cadmium water,and alpha-lipoic acid was gavaged daily on time.In order to reduce the error,other groups of rats were also gavaged to DIW.During the experimental period,the daily body weight,water intake and food intake of rats were recorded.The samples were collected after 12 weeks,and then the oxidative performance indicator kit,histology and Western blot were used to detect changes in oxidative damage,PARP-1 protein expression and apoptosis-related indexes in the kidney.The results showed that exposure to 50 mg/L of cadmium for 12 weeks would induce oxidative damage in rat kidney cells and promote the expression of PARP-1 and AIF,while activating the mitochondrial apoptotic pathway;antioxidant a-lipoic acid could relieve the oxidative damage caused by cadmium,and significantly reduce the expression of PARP-1 and apoptosis(P<0.05 or P<0.01).By analyzing the above experimental results,the following conclusions can be drawn,①Cadmium exposure can induce oxidative damage and apoptosis of rat renal tubular epithelial cells,and activate PARP-1-dependent Parthanatos.Besides,Parthanatos activation will further aggravate oxidative damage and apoptosis.②When DNA damage caused by cadmium was mild,PARP-1 could repair DNA damage and inhibit cell cycle arrest.When DNA damage is severe,excessive activation of PARP-1 promotes cell cycle arrest and induces apoptosis and Parthanatos.③After activation of the ERK1/2,on the one hand,it will promote the expression of PARP-1 protein,and on the other hand,it will suppresse apoptosis by up-regulating autophagy.④Cadmium exposure will cause oxidative damage to the renal cortex of rats,and adding antioxidant intervention during this process can alleviate renal cortical damage and inhibit PARP-1 protein expression as well as apoptosis.