The Intervention And Mechanism Of Potentilla Anserin Flavonoids For Oxidative Toxicity Mediated Osteoporosis In Cadmium

In recent years,oxidative stress has been highly valued as a risk factor for osteoporosis?OP?.Due to its toxicity,the heavy metal cadmium?Cd?can lead to the imbalance of intracellular ion homeostasis,the damage of antioxidant system and mitochondrial,thus leading to the increase of ROS.Increasing ROS can lead to oxidative stress,bone density decreased and bone loss.Therefore,oxidative stress is the main cause of OP.The main mechanism of oxidative stress leading to osteoporosis is imbalance of ion homeostasis,mitochondrial injury,activation of cysteinyl aspartate-specific pathways,leading to apoptosis.Osteoprotegerin?OPG?expression was decreased,Receptor Activator of Nuclear Factor-?B Ligand?RANKL?expression was up-regulated,osteoclast was activated and bone resorption was promoted.Therefore,the application of antioxidant-active substances may be a new target for the prevention and treatment of OP.Most flavonoids have phenolic hydroxyl structure,which can form stable chelates with metal ions,prevent metal ions from catalyzing the generation of oxygen free radicals,and terminate lipid peroxidation.It also can be used as the receptor to hamper the free radical chain reaction and thus act as an antioxidant.It has been reported that Potentilla anserin flavonoids?PAF?has strong anti-lipid peroxidation and inhibition of free radical production on edible oils,and the anti-free radical ability is better than that of vitamin C and citric acid.This group putforward the hypothesis that PAF play an anti-OP role by reduceing the excessive accumulation of ROS caused by Cd poisoning,inhibiting oxidative stress,and then causing FoxO signal path reduction,and stimulating the activation of the Wnt pathway.Purpose:Based on the above results,this study simulated the oxidative stress of bone cells and OP of oxidative stress in animals,and established the model of Cd infected cells and OP animals,with ROS increasing as the cause of OP and the mechanism of oxidative stress leading to OP.Under the intervention of gradient concentration of PAF and NAC,the activity of PAF and its mechanism were investigated through studies on oxidation,anti-apoptosis,related protein expression,bone and its biomechanics.It provides experimental basis for further understanding the pathogenesis of OP,the bone oxidation toxicity of Cd and the prevention and treatment of OP,and also provides ideas for the development and utilization of Potentilla anserine L.Method:1.Microwave assisted ethanol extraction method for the extraction of PAF from qinghai yushu autumn Potentilla anserine L.2.MC3T3-E1 cells and BALB/c male mice were poisoned with Cd in gradient concentration,and the cell and OP animal model of Cd cytotoxicity was established,mainly in bone mineral density,bone tissue morphology measurement,the change of bone biomechanics for OP model evaluation index,so as to determine Cd modeling dose.3.Antioxidant capacity of PAF was measured by in vitro antioxidant assay and MC3T3-E1 cell culture assay.4.Using microscopes and transmission mirrors,colorimetry,CCK-8 and flow cytometry,biochemical method,ELISA method,western Blot and qRT-PCR to observe and detect reactive oxygen species?ROS?,glutathione peroxidase?GSH-Px?and superoxide dismutase?SOD?,catalase?CAT?activity,malondialdehyde?MDA?content and phosphating p66^shc protein expression;alkaline phosphatase?ALP?,bone gla Protein?BGP?levels,OPG,RANKL and human anti-lilac acid phosphatase?TRACP5b?levels.Cell survival and apoptosis,intracellular free calcium ion concentration([Ca²⁺]i),mitochondrial membrane potential?MMP?,apoptosis-related proteins,bone and fat differentiation regulators,and expression levels of related genes and proteins in FoxO3a and Wnt signaling pathways.5.The changes of BMD,bone tissue morphology and bone biomechanics were detected.Result:1.Microwave-assisted ethanol extraction was used to extract PAF qinghai yushu autumn Potentilla anserine L.The optimum conditions were:ethanol concentration60%,solid-liquid ratio 1:25,microwave time 20 min,microwave temperature 70?.Extraction:14.63±0.21 mg/g.2.In vitro,PAF showed antioxidant activity at the concentration of 3.125 g/mL,while the 25 g/mL in cells was significant,with an obvious dose-effect relationship.3.The final concentration of MC3T3-E1 cells infected with Cd gradient concentration reached 30?M,with the highest ROS content.The concentration of Cd gradient concentration infected with BALB/c male mice was up to 2 mg/kg,and there were changes in bone density,bone tissue morphology and bone biomechanics.4.PAF intervention can increase GSH-Px,SOD and CAT in Cd infected cells and osteoporosis animal models,and reduce ROS,MDA content and phosphating p66^shchc protein expression.The concentration of[Ca²⁺]i decreased significantly,MMP recovered,Na⁺-K⁺-ATPase,Na⁺-K⁺-ATPase and ATPase activity increased,Bcl-2expression increased,Bax expression decreased,Bcl-2/Bax ratio increased,Cytochrome C?CytC?,apoptosis-inducing factor?AIF?,Cleaved caspase-3 protein expression decreased,cell survival rate increased,apoptosis rate decreased.ALP,BGP,OPG and OPG/RANKL were increased,and the expression of RANKL and TRACP5b were decreased.Improvebone differentiation,reduce the expression of pronouns associated with fat differentiation,reduce the expression of FoxO3a protein and Gadd45?genes,and improve the expression of?-catenin protein and target gene Axin2in the Wnt signaling pathway.5.PAF intervention can significantly increased BMD,bone trabecular thickness,trabecular bone volume fraction,and the bone trabecular spacing was decreased,while Maximal Loading and Elastic Modulus increased significantly.Conclusion:1.For the first time,the Potentilla anserine L.in qinghai was extracted by microwave-assisted ethanol extraction,and the optimal extraction amount was 14.63±0.21mg/g?2.Initial use of Cd oxidation toxicity to establish the animal model of OP.Cd toxic dose?Cell:CdCl₂ 30?M;Mouse:CdCl₂ 2mg/kg?can be used to establish cytotoxicity and OP animal models,respectively.3.PAF was non-toxic,effective antioxidant dose:Cell 6.25²5mg/L and Mice 1.5mg/kg.4.The bone toxicity of Cd results from oxidative stress,imbalance of[Ca²⁺]i homeostasis and apoptosis.PAF can resist oxidation,stabilize ion balance and protect bone cell activity.5.Cd decreases OPG and increases RANKL through RANKL/RANK/OPG signaling pathway,promotes bone resorption,and leads to OP.PAF has better antioxidant,stable ion balance,protect bone cell activity,inhibit bone absorption,regulate bone absorption and bone production balance,to achieve the role of anti-osteoporosis.6.The bone toxicity in Cd promotes OP occurrence by inhibiting the Wnt/?-catenin signaling pathway responsible for bone differentiation,activating FoxO3a transcription factors and target genes,inducing oxidative stress and inhibiting bone formation.PAF can play anti-OP role by regulating The FoxO3a/Wnt signal ingresss.