Protective Effect Of Melatonin In Cadmium-induced Apoptosis In Rat Proximal Tubular Cells

Cadmium(Cd),is an occupational hazard and environmental pollutant which can enter the body through digestive tract and respiratory tract with slow metabolism and long biological half-life.Accumulation of Cd cause toxic damage to a variety of tissues and organs.The kidney is the main target organ for the toxic effects of Cd,in which the proximal tubular is the main target site.Long-term Cd exposure causes proximal tubular reabsorption dysfunction,and eventually leads to renal failure.Melatonin(Mel)is an endogenous hormone secreted mainly from the pineal gland regulated by circadian rhythm,which has a series of biological activities such as anti-oxidation,anti-inflammatory,and anti-apoptosis with few side effects,economic safety.Thus,Mel is widely applied in the treatment of clinical cardiovascular disease,diabetes and neuroendocrine related diseases and its complications.Mitochondria are important organelles in eukaryotic cells,and mitochondrial fission plays the key role in mitochondrial homeostasis.Studies have shown that the protective effect of Mel on cardiovascular disease,diabetes and its complications is closely related to mitochondrial function.However,the role of Mel and mitochondrial fission in Cd-induced renal toxicity is largely unknown.Based on the above understanding,this study used rat proximal tubular cell line and primary rat proximal tubular cells as materials to explore the effects of SIRT1/PGC1αsignaling pathway,mitochondrial fission and apoptosis in Cd-induced rat proximal tubular cells injury and to explore whether Mel plays a protective effect in Cd-induced rat proximal tubular cells injury by relieving abnormal mitochondrial fission and apoptosis and to provide a basis for the prevention and treatment of cadmium poisoning in the clinic.1.Effect of cadmium on mitochondrial fission and apoptosis of rat proximal tubular cellsThis study was carried out on rat proximal tubular cell line(NRK-52E cells)and primary rat proximal tubular cells(rPT cells).The cells were treated with different concentrations of Cd(0,1.25,2.5,5 μM)for 12 hours.ROS level was detected by the DCFH-DA staining.Expression of SIRT1,PGC-1α and mitochondrial fission-related genes and proteins were detected by QRT-PCR and Western blot,respectively.Mitochondrial network structure was observed by laser scanning confocal microscope(LSCM),Nuclear morphology was checked with DAPI staining by LSCM,and the apoptosis rate was detected by flow cytometry(FCM).The results showed that compared with the control group,ROS level increased significantly(P<0.01)in Cd treatment group.Genes and proteins expression of SIRT1,PGC-1α were significantly reduced(P<0.05 or P<0.01).Gene expression of Drp1 showed no significant change,but protein expression was significantly increased(P<0.05 or P<0.01).Gene and protein expression of Fis1 were significantly increased(P<0.05 or P<0.01).Mitochondrial network was broken,the fragments were scattered and distributed.Nuclear morphology of some cells showed that the chromatin distribution was uneven,and the nucleus appeared shrinkage,dissolution,and depression and other apoptotic morphological characteristics and the apoptosis rate was significantly increased(P<0.05 or P<0.01).These results indicated that long-term treatment with low concentration of Cd may induce apoptosis of rat proximal tubular cells by inhibiting the SIRT1/PGC1α signaling pathway and promoting abnormal mitochondrial fission.2.Role of melatonin on cadmium-induced mitochondrial fission and apoptosis of rat proximal tubular cellsThe rat proximal tubular cells were treated with 2.5 μM Cd in presence or absence of 10μM Mel pretreatment for 2 hours.Cell viability was detected by CCK-8 assay,cell morphology was observed by phase contrast microscope,ROS level was detected by the DCFH-DA staining.Expression of SIRT1,PGC-1α and mitochondrial fission-related genes and proteins were detected by QRT-PCR and Western blot,respectively.Mitochondrial network structure was observed by LSCM.Nuclear morphology was checked by LSCM,and the apoptosis rate was detected by FCM.The results showed that compared to the control group,cell viability was no significant change in 10～200 μM melatonin pretreatment for 2 hours,and compared to Cd treatment group,cell viability was significantly increased(P<0.01),cell density increased,shrinkage cells decreased,and ROS level significantly decreased(P<0.05 or P<0.01).Genes and proteins expression of SIRT1,PGC-1α were significantly increased(P<0.05 or P<0.01).Genes and proteins expression of Drp1,Fis1 were significantly reduced(P<0.05 or P<0.01).The damage of the mitochondrial network structure was significantly relieved,and the degree of fragmentation was reduced in Cd and Mel co-treatment group.Apoptosis of rat proximal tubular cells was decreased which presented to be more normal nuclear morphology,and the apoptosis rate is significantly reduced(P<0.05 or P<0.01).These results indicated that Mel could improve the an-tioxidant capacity of cells by eliminating ROS and alleviate oxidative stress,and may inhibit abnormal mitochondrial fission through the SIRT1/PGC-1α signaling pathway,thereby play a protective effect on Cd-induced rat proximal tubular cells injuryConclusions:long-term treatment with low concentration of Cd could increase the ROS level and promote abnormal mitochondrial fission of the rat proximal tubular cells.Mitochondrial fission can be negatively regulated by SIRT1/PGC-1α and participate in the apoptosis of rat proximal tubular cells induced by Cd.Mel could improve the antioxidant capacity of the rat proximal tubular cells by eliminating ROS and alleviate oxidative stress.Mel could inhibit abnormal mitochondrial fission through the SIRT1/PGC-1α signaling pathway,and reduce the apoptosis of rat proximal tubular cells induced by Cd.