Protective Effect And Mechanism Of Potentilla Anserina Polysaccharides On Cadmium Induced Nephrotoxicity

BackgroundCadmium(Cd)is a toxic heavy metal existing ubiquitously in soil,water and air.Cd in the environment couldn't be metabolized by creatures that results in Cd accumulation in vivo through the food chain.Because of the similar properties with zinc,Cd2+ is easy to replace Ca2+,Zn2+,Mg2+ in vivo and cause diseases.The level of impairment depends on the Cd dose,the form of Cd and its exposure duration in organism.Cd in blood can be delivered throughout the whole body,especially accumulating in kidney,liver,spleen,thymus and bone.Due to the renal reabsorption,Cd accumulates constantly in renal tubules which leads to cell damage,mitochondrial dysfunction and apoptosis.Heavy metal chelating agent and antioxidant treatment are currently the major therapy for Cd toxication.Researches showed that some extract of Chinese herbal medicines possessed capability against Cd induced nephrotoxicity and impairment.Potentilla anserina L is used as food as well as a traditional medicine which contains abundant nutrients.More attention has been paid on Potentilla anserina polysaccharides(PAP)because of its specific biological activity.Researches showed that PAP had effect on anti-oxidant,anti-apoptosis and immunoregulation both in vivo and in vitro experiments.ObjectiveThe activities of PAP including anti-oxidation,anti-apoptosis and immunoregulation are targeted to Cd induced damage.In this research,Cd exposed models including cells and mice will be established.The Cd distribution and content in cells and mice,PAP protective effect and related mechanism on Cd induced cytotoxicity and nephrotoxicity will be investigated.Methods1.PAP was obtained by water extraction,ethanol precipitation and deproteinization.Infrared spectroscopic analysis(IR),phenol-sulfuric acid method and high performance liquid chromatography(HPLC)were performed for PAP analysis.2.Cd exposed models including cells and mice were established,fluorescent probes were synthesized and used to label Cd2+ in HEK293 cells and mice organs.Cd2+ distribution in mice organs was imaged and quantified by laser-scanning confocal microscope.The pathological alteration of each organ in mice was determined by H&E staining.3.PAP was used in Cd exposed HEK293 cell model.The CCK-8 method was used to detect cell viability.Fluorescent staining was used to analyze the cell apoptosis,mitochondrial membrane potential and ROS level.ELISA method was used to determine GSH,SOD,MDA,ATP level in cells.Flow cytometry was used to investigate the apoptosis ratio.Western blotting method was used to analyze the expression of mitochondrial generation and function related proteins,apoptosis related proteins involving intrinsic apoptotic pathway and extrinsic pathway.4.The acute and chronic Cd exposed mice models were established and PAP was supplied.Immunohistochemistry staining and Masson's staining method were used for histopathological examination and fibrosis progression.Other methods mentioned above were used to investigate renal function,redox status,expression of mitochondrial function and generation related proteins,intrinsic and extrinsic apoptotic pathway related proteins.Results1.The polysaccharides content of Potentilla anserina L from Yushu in Qinghai province was 64.22%.PAP contained characteristic structures of saccharides.PAP was mainly comprised of Glu,Rha,Gal-UA,Gal-N,Glc,Gal,Xyl and Ara.2.Fluorescent probes labeled the dispersive distribution of Cd2+ in cytoplasm,concentrated accmuluation was also observed in some specific organelles.Cd2+ distribution pattern and quantification in each organ was exhibited by the fluorescent probe staining.The Cd2+ accumulation in kidney was predominant especially in renal tubular epithelial cells,and then was in liver,spleen,thymus and testis.3.PAP pretreatment could improve cell viability and redcue cell apoptosis in Cd treated cells.The GSH and SOD activities were elevated,and the ROS and MDA content in cells were reduced after PAP pretreatment.The depressed mitochondrial membrane potential and ATP content were reversed,and the expression of PGC-1? and Nrf2 were regulated by the PAP pretreatment.The flow cytometry showed that the apoptosis ratio in Cd treated cells was reduced after PAP pretreatment.Western blotting data demonstrated that the bcl-2 expression and bcl-2/bax ratio were up-regulated,and the expression of ?H2ax,p-p53,cytochrome C,Cleaved-Caspase 3 and Fas proteins were down-regulated by the PAP pretreatment.The underlying mechanism related to anti-apoptosis was involved in regulating both mitochondrial dependent intrinsic apoptotic pathway and death receptor-initiated extrinsic pathway.4.After PAP pretreatment,kidney index and renal function were improved in acute and chorinc Cd exposed mice.The GSH activity in kidney was increased,and MDA content was decreased.The expression of PGC-1? and Nrf2 were regulated in both acute and chronic Cd exposed mice.In addition,renal histopathology and fibrosis progression were alleviated by PAP pretreatment.The bcl-2/bax ratio was up-regulated,and Fas expression was down-regulated after pretreatment.The mechanism related to anti-apoptosis in animal experiments was also involved in regulating both intrinsic and extrinsic apoptotic pathway.ConclusionPAP pretreatment could improve cell viability,renal function,mitochondrial integrality and function.The Cd induced nuclear DNA damage,renal histopathological changes were also alleviated by PAP pretreatment.PAP exhibited obviously anti-oxidative and anti-apoptosis capability on Cd induced nephrotoxicity.