Protective Effect Of Puerarin In Cadmium-induced Mitophagy And Apoptosis In Rat Proximal Tubular Cells

Cadmium(Cd),a common heavy metal eontaminant in the environment,can cause damage to a variety of organs and tissues.In recent years,with the rapid increase of cadmium production and usage and serious industrial pollution,the cadmium content in the environnent has risen rapidly,and public health hazards caused by cadmium exposure have caused widespread concern.The kidney is one of the most important target organs for cadmium toxicity.Among them,the renal tubule is the main target site for toxic damage of cadmium.Long-term exposure to cadmium in the body will cause irreversible damage to the kidney.Puerarin(PU)is a kind of plant isoflavone extracted from the dried root of Chinese medicinal kudzu.It has anti-oxidation,micro-circulation,hypoglycemic and other pharmacological functions,and has the characteristics of low toxicity,fast metabolism,safety and reliability,which makes it widely used in the treatment of cardiovascular diseases and diabetes,complications,etc.in the clinic.Mitochondria are important bilayer membrane organelles,in addition to providing energy necessary for the normal activity of the cells,but also participate in the process of cell differentiation,cell signaling and apoptosis,etc.In addition,Mitophagy is considered to be the main pathway for mitochondrial quality and quantity control.At present,puerarin and mitophagy are less studied in the process of cadmium-induced nephrotoxicity.In this study,primary rat proximal tubular cells were used as a model to study how puerarin exerts protective effects on cadmium-induced apoptosis by regulating mitophagy,and provide new ideas for preventing toxic effects of cadmium on the kidney.1.Effect of cadmium on apoptosis of rat primary renal proximal tubular cellsThis study was carried out on primary rat proximal tubular(rPT)cells cultured with mechanical gridding,filtering and chemical digestive methods.The first passage of rPT cells at logarithmic growth phase were used to perform the experimental design.rPT cells were treated with different concentrations of cadmium(0,1.25,2.5 jaM)for 12 h.and the following experiments were performed.Cells viability was measured with CCK-8 assay.Nuclear morphology was checked with DAPI staining,and apoptosis of rPT cells detected by flow cytometry(FCM).The results showed that,Cd treatment group compared to the control group,cells viability was significantly decreased(P<0.05 or P<0.01).Nuclear morphology of some cells appear crenulation and depression and the apoptosis rate was significantly increased(P<0.05 or P<0.01),and expression of Cleaved caspase-3 was also significantly increased(P<0.05 or P<0.01).It indicated that long-term treatment of low concentration cadmium can cause the decrease of rPT cells viability and lead to apoptosis.2.Effect of cadmium on mitophagy of primary rat proximal tubular cellsrPT cells were treated with different concentrations of cadmium(0,1.25,2.5 ?M)for 12 h.and the following experiments were performed.Mitochondrial membrane potential(MMP)was detected by JC-1 staining,ROS level was determined by DCFH-DA staining and ATP level was measured with luciferase assay.Mitochondrial ultrastructure and the number of lysosomes devouring mitochondria were checked with transmission electron microscopy(TEM),mitochondrial stained with Mito-Tracker Red and lysosome stained with Lyso-Tracker Green were observed with laser scanning confocal microscope(LSCM)to check reticulum structure and co-location.Expression of mitophagy-related genes and proteins were detected by QRT-PCR and westerm blot,respectively.The results showed that,Cd treatment group compared to the control group,ATP content and MMP were significantly decreased(P<0.05 or P<0.01)?and the ROS content was significantly increased(P<0.05 or P<0.01).Mitochondrial networks appear fragmented,mitochondrial raft rapture,membrane disruption and matrix ambiguity were observed,co-location level of mitochondria and lysosome was increased.Genes and proteins expression level of DRP1,PINK1 and Parkin were significantly increased(P<0.05 or P<0.01).These convincing data manifest that long-term treatment of low concentration cadmium could damage the mitochondrial structure and function of rPT cells and lead to the occurrence of mitophagy.3.Effect of mitophagy on apoptosis in primary rat proximal tubular cells induced by cadmiumrPT cells were treated with 2.5 uM cadmium in presence or absence with mitochondrial division inhibitor Mdivi or PINK1 siRNA for 12 h,and the following experiments were performed.Expression of mitophagy-related proteins and Cleaved caspase-3 protein were detected by western blot,and apoptosis of rPT cells detected by flow cytometry(FCM).The results showed that,Mdivi or PINK1 siRNA and Cd co-treatment group compared to Cd treatment group,Expression level of PINK1 and Parkin were significantly decreased(P<0.05 or P?0.01).Expression of Cleaved caspase-3 and apoptosis rate were significantly decreased(P<0.05 or P<0.01).It is shown that inhibition of mitophagy can block Cd-induced apoptosis of rPT cells.4.Effect of puerarin on cadmium-induced mitophagy and apoptosisin primary rat proximal tubular cellsrPT cells were treated with 2.5 ?M cadmium in presence or absence with 50 ?M puerarin for 12 h,and the following experiments were performed.Cells viability was measured with CCK-8 assay,MMP was detected by JC-1 staining,ROS level was determined by DCFH-DA staining and ATP level was measured with Iuciferase assay.Mitochondrial ultrastructure and the number of lysosomes devouring mitochondria were checked with transmission electron microscopy(TEM),mitochondrial stained with Mito-Tracker Red and lysosome stained with Lyso-Tracker Green were observed with laser scanning confocal microscope(LSCM)to check reticulum structure and co-location.Expression of mitophagy-related genes and proteins were detected by QRT-PCR and western blot,respectively.Nuclear morphology was checked with DAPI staining,and apoptosis of rPT cells detected by flow cytometry(FCM).The results showed that,PU and Cd co-treatment group compared to Cd treatment group,cells viability was enhanced significantly(P<0.05),MMP and ATP level were restored(P<0.05),and ROS level was down-regulated significantly(P<0.05).The damage of mitochondrial networks and mitochondrial ultrastructure were alleviated dramatically,co-location level of mitochondria and lysosome was decreased.Genes and proteins expression level of DRJP1,PINK1 and Parkin were decreased significantly(P<0.05 or,P<0.01).Apoptosis of rPT cells was decreased which presented as more regular nuclear morphology,decreased expression of cleaved caspase-3 and apoptosis rate(P<0.05 or P<0.01).These convincing data manifest that PU prevents Cd-induced apoptosis of rPT cells via inhibiting mitophagy.