The Effect Of Potentilla Anserina Polysaccharides On Cadmium Induced Apoptosis And Autophagy In Nerve Cell

The effective components of chinese traditional medicine have effects of antioxidant,anti-apoptosis and inhibition of cell autophagy,and their effects are ensure cell survival,such as?-Asarone,Salvianolic acid B,Ginsenoside Rg1 and Ginsenoside Rb1,etc.Potentilla anserine has the effect of nourishing and strengthening life,invigorating the spleen and reinforcing stomach,invigorating qi and blood,nourishing the Yin and nourishing the kidney.Potentilla anserine polysaccharide?PAP?,the main active component of the traditional Tibetan medicine potentilla anserine,has been proved to have the effects on antioxidant and enhancing immunity.It is very necessary that explore the neurotoxicity Cadmium?Cd?and protection of PAP,taking the antioxidant effect of PAP as a point of penetration.Cd is a toxic heavy metal that enters the body through contact with the digestive tract,respiratory tract or skin and becomes an exogenous chemical toxic substance.Because of its long half-life?10²0 years?,it is easy to accumulate in various tissues and organs in the body.The toxicity of Cd is related to its physical and chemical properties.Besides the long half-life,the toxicity of Cd is also related to its ion radius?97 pm?,which that very similar to the radius of Ca²⁺?99 pm?.Cd²⁺can replace the binding site or ion channel of Ca²⁺,and can also damage tissue cells and cause persistent pathological changes through compete binding site or ion channel of protein molecules with Zn²⁺,Fe²⁺.Neurotoxicity refers to damage of the structure and function in the central nervous system and peripheral nerves caused by exogenous chemicals.In first,Cd affects the integrity and permeability of the vascular endothelium,and more Cd enters the central nervous system and accumulates long time induce to changes in the structure of nerve cells and glial cells,mitochondrial damage,DNA breakage,cell death,etc.The mechanism of Cd neurotoxicity has been reported,most of which was based on apoptosis induced by Cd through the study of cultured cells in vitro.It is believed that the neurotoxicity of Cd mainly derive from its oxidative stress response and intracellular calcium dyshomeostasis.Oxidative stress leads to lipid peroxidation,and lipid peroxidation of mitochondrial bilayer membrane leads to changes in mitochondrial membrane structure and function,such as mitochondrial membrane potential decrease,so mitochondria must be the target organ of Cd toxicity.Cd competitive inhibition Ca²⁺through the Voltage-dependent gated channels,switch state of voltage dependent anion channel?VDAC?,which influence the flow efficiency of metabolites molecules and ions inside and outside of mitochondria membrane.When VDAC open,Ca²⁺the VDAC enter into the mitochondria easily,resulting in mitochondria swelling and apoptosis.To sum up,the neurotoxic effects of Cd is mainly due to the oxidative stress and competitive inhibition of Ca²⁺channels induced by Cd,and affect the mitochondrial membrane and the flow of its ions.The traditional Tibetan medicine potentilla anserine,has been proved to have a protective effect on brain hypoxic damage caused by various factors,and its mechanism is closely related to the elimination of free radicals,inhibition of oxidative stress damage and improvement of mitochondrial function.According to traditional medicine and modern pharmacology analysis,potentilla anserine have the potential to be become a new drug to fight against Cd.Aims:PAP,the main active ingredient of potentilla anserine,for the first time,was used for to intervene with Cd infected nerve cells and mice.The mechanism of Cd neurotoxic effects on the central nervous systems and possible protective effect of PAP were discussed,and providing theoretical basis for further understanding of neurotoxic effect of Cd,prevention and treatment of Cd poisoning,and development and utilization of potentilla anserine.Methods:1.Mouse neuroblastoma N2a cell line?N2a?,human neuroblastoma cell line SH-SY5Y and BALB/c mice were selected as experimental subjects.2.The model of cell and mouse were established by Cd gradient concentration,and the toxic doses of Cd was determined.,and selection and determination of the most effective dose by PAP gradient concentration drug administration.3.To measure brain body coefficient and observe the general changes of brain and other organs in animals.The morphological changes of cultured nerve cells and animal brain cells were observed microscopically and microscopically.Cellular viability of cells was assessed by CCK-8 assay.Fluorescence and flow cytometry was used to evaluate the apoptotic effect on nuclear morphology,apoptosis,fluorescent intensity of ROS?mitochondrial membrane potential?MMP?and intracellular calcium ion concentration([Ca²⁺]i).Acidic vesicular organelles?AVOs?and autolysosome were detected by acridine orange?AO?and Monodansylcadaverine?MDC?staining,respectively.The activity of SOD,CAT,MDA,H₂O₂,Na⁺K⁺-ATPase,Ca²⁺Mg²⁺-ATPase and total ATPase were detected by colorimetric analysis.The expression of apoptotic related proteins and associated protein of apoptosis signal pathway was detected by western blotting?immunohistochemistry and immunofluorescence analysis.Result:1.Cd infected cells and animals showed increased apoptosis and mortality,except for neurological symptoms and changes in organ and brain coefficients,and pretreatment with PAP reduce symptoms of infected animals,injury of liver and spleen,brain body coefficient,cell apoptosis and mortality reduction.2.The neurotoxic effects of Cd are related to oxidative stress.ROS levels induced by Cd were significantly increased,SOD activity were significantly decreased,CAT activity,content of MDA and H₂O₂ were markedly elevated,pretreatment with PAP reversed Cd induced the above events,inhibits oxidative stress caused by Cd neurotoxicity.3.An significant increase in[Ca²⁺]i were observed in Cd infected cells and animals,and phosphorylation of CaMK II activate Akt/mTOR signal transduction pathway.Pretreatment with PAP reduce phosphorylation levels of CaMK II,which in turn stops activation of Akt/mTOR signal transduction pathway and inhibits apoptosis.4.The morphologic change of nuclei was observed to show,shrinkage and pyknosis or karyorrhexis,and mitochondria swelling,decrease or disappearance of mitochondria crista,some mitochondrial vacuolation appeared in Cd infected cells and animals.An significant decreases in the activity of Na⁺K⁺-ATPase?Ca²⁺Mg²⁺-ATPase and total ATPase and loss of MMP were observed.The ratio of Bcl-2/Bax was reduced significantly,up expression of AIF,Cyt C,Cleaved caspase-3 and Cleaved PARP were correlated with Caspase-dependent and non-caspase-dependent apoptosis pathways.Pretreatment with PAP reversed Cd induced the above events,inhibits the activation of Caspase-dependent and non-caspase-dependent apoptosis pathways.5.A large number of autophagosome can be seen in the ultrastructure of Cd infected cells and animals brain cell.The increase red fluoresent AVOs were found by AO,and MDC fluorescence accumulation spots were increased significantly that were absent in control cells.The number of FJB positive cells in the cerebral cortex and hippocampal areas of the Cd group was significantly increased with strong staining intensity.The level of PI3K class III,Beclin-1and LC3 II proteins were increased,and the level of p62 proteins were increased.Immunofluorescence was performed to observe the expression and distribution of LC3,showed that LC3 punctate witch localizated in around of the nuclear in Cd group were significantly more than in control group,and CA3 region>Hilus region>CA1 region in hippocampal areas were presented.Pretreatment with PAP could significantly reduce AVOs formation and improve cytoplasmic cavitation,increased expression of Bcl-2 and p62,decreased protein levels of PI3K class III,beclin-1 and LC3 II,and inhibited the conversion of LC3 I to LC3 II.Combined use of autophagy inhibitor LY294002 was more effective.Conclusion:1.Cd has toxic effects on nerve cells and brain cells of mice,and PAP can antagonize the neurotoxicity induced by Cd.2.Cd neurotoxicity results from oxidative stress and imbalance of intracellular calcium homeostasis.PAP can inhibit oxidative stress and stabilize intracellular calcium homeostasis.3.Cd neurotoxicity is dominated by apoptosis and autophagy death.The protective effect of PAP is closely related to inhibition of apoptosis and autophagic cell death.4.Cd neurotoxicity induce the nerve cell apoptosis increased and autophagic cell death,inducing caspase-dependent apoptotic pathways and caspase-independent apoptotic pathway.Activated Akt/mTOR signaling pathway by phosphorylation of CaMKII?The antiapoptotic effect of PAP is related to stabilizing intracellular calcium ion balance and protecting mitochondrial membrane?5.The pathway of Cd neurotoxicity induced autophagy may be the PI3K class III/beclin-1 signaling pathway,and PAP play a role in preventing autophagic cell death by regulating the expression of autophagy related factors.