Role Of Mitochondrial Pathway In The Protective Effect Of Puerarin Against Lead-induced Apoptosis In Primary Cultures Of Rat Proximal Tubular Cells

Lead(Pb) is a common environmental and occupational heavy metal pollutant. With the rapid development of our industry and agriculture, the ecological environment is being widely polluted by Pb. The Pb in environment cannot be biodegradable and it can enter into body through many ways and cause serious damage to human health. Pb is chronic accumulated poison. If low dose of Pb is continued ingested, it would be accumulated in body with a toxic effect. As a multi-organ toxicant, lead exerts potent toxic effects on different tissues including the liver, heart, kidney, brain and the hematopoietic system. Kidney is a sensitive target organ for lead exposure, of which proximal tubular epithelium is the main site of Pb-induced nephrotoxicity. Puerarin(PU) is an isoflavone isolated from a traditional Chinese medicine ge-gen. Many studies have found that puerarin has anti-bacterial and anti-inflammatory properties which could improve immune function; Meanwhile, Puerarin is an effective natural free radical scavenger which can prevent cells from DNA damage and oxidative stress injury; In addition, some evidence shows that PU can alleviate renal damage induced by nephrotoxins. Our previous study has demonstrated that the apoptotic death was the chief mechanism in Pb-induced nephrotoxicity in rPT cells. Mitochondria, as a central part of the intrinsic apoptosis pathway, plays a chief role in the regulation of apoptosis. Animal experiments have confirmed puerarin had significantly protective effects on lead-induced nephrotoxicity. But it has not yet been thoroughly clarified whether puerarin has a protective effect on lead-induced apoptosis through mitochondrial pathway in rPT cells. In this study, rPT cells were incubated with Pb and/or PU for 12 h to determine the optimal concentration for its protective effect by evaluating the cell viability, morphological investigation using phase-contrast microscopy, the apoptotic rates and ROS production measured by flow cytometry as well as the level of intracellular MDA. Confocal laser scanning microscopy and immunoblotting technique were employed to detect the openning of mitochondrial permeability transition pore(MPTP) and analyse the release of apoptotic factors(cytochrome c) and the expression of apoptosis key enzyme(caspase-9, caspase-3 and PARP), respectively. Meanwhile, chemiluminescence method was used to detect the level of ATP in the cells, then we analysed the expression of ATP transporter and changes in mitochondrial morphology. In addition, the levels of Bcl-2 and Bax gene transcription and protein translation were detected. The results showed that 100 ?M Puerarin had a significant protective effect on lead-induced damage in rPT cells by decreasing the apoptosis rate and restoring cell morphology and nomal intracellular levels of ROS and MDA; Meanwhile, Pb-mediated mitochondrial permeability transition pore opening together with mitochondrial cytochrome c release, activations of caspase-9 and caspase-3, and subsequent poly ADP-ribose polymerase(PARP) cleavage can be effectively blocked by the addition of PU; PU can reverse Pb-induced ATP depletion by restoring mitochondrial membrane potential and regulating the ATP transporter expression levels of ANT-1 and ANT-2; In addition, up- and down-regulations of Bcl-2 and Bax with increased Bcl-2/Bax ratio due to PU administration further alleviated Pb-induced mitochondrial apoptosis. In summary, PU may play it anti-apoptotic ability through affecting MPTP opening and subsequent apoptotic events against mitochondrial damage resulted from lead exposure. PU reversed Pb-induced ATP depletion by reversing MMP collapse, regulating ANT isoforms expression and restoring mitochondrial dynamic network.