Metabolism Of Mulberry Anthocyanins And Its Mechanism On Nutrition Intervention To Nerve Cell Damage Inducded By Lead

Lead?Pb?is a kind of heavy metal,commonly found in the natural environment with strong toxicity.Pb can accumulate in the body and cause multiple organ toxicity.In particular,low-level Pb exposure with very strong neurotoxicity can cause nerve cell damage and consequent irreversible damage to children's cognition,learning and memory ability.The effects of Pb have attracted extensive attention lately.Mulberry?Morus alba L.?fruit,has long been served as a multifunctional Chinese medicine to nourish the kidneys and blood,and to cure anemia,dizziness or fatigue,because of the abundant source of active ingredient,especially anthocyanins?ACNs?.Nutritional intervention is an effective method to prevent lead poisoning.ACNs are natural polyphenol pigments that widely distribut in plant-based diet.They can penetrate the blood brain barrier and possess nerve protective effect.The project is to study the metabolism regularities of ACN monomers in vitro and in vivo,and evaluate the effects of ACN monomers on learning and memory function in Pb²⁺exposed rats,obtain the active ingredient with the good ability to protect the nerve cells lesion.The intervention mechanism of the active ingredient will be further studied from the molecular level through the expression of cognitive proteins and genes in the cAMP-PKA-CREB signaling pathways.The investigation will clarify the molecular mechanism on intervention effect to Pb²⁺-induced nerve cells lesion by active ingredient and confirm the key factor in intervention process.The project will provide a scientific basis for the application of the mulberry ACNs,and prevention the lead poisoning by nutrition intervention.The experimental procedures and results of the study are presented as below:?1?Column chromatography combined with HSCCC was employed to isolate high-purity ACN monomers from mulberry fruits.Three high-purity anthocyanin monomers of Delphinidin-3-O-rutinoside?D3R?,Cyanidin-3-O-rutinoside?C3R?,and Cyanidin-3-O-glucoside?C3G?were obtained,and identified by NMR and HPLC-ESI-MS/MS.?2?The gastric-intestinal digestion and the bacterial-dependent metabolisms of C3G,C3R,D3R were investigated in vitro.ACNs and metabolites were analyzed by HPLC-ESI-MS/MS.In the gastric-digestive system,a small amount of methylated-ACNs occurred.In the intestinal digestive system,C3G and C3R mainly decomposed into protocatechuic acid?PCA?,and2,4,6-trihydroxybenzaldehyde,D3Rmainlydecomposedintogallicacidand2,4,6-trihydroxybenzaldehyde.In the bacterial system,C3G,C3R,D3R showed fast degradation rate at the first 4 h.C3G disappeared after 6 h of metabolism,while C3R and D3R were no longer detected after 8 h.The metabolism of C3G and C3R mainly resulted in the formation of PCA,vanillic,p-coumaric acid and 2,4,6-trihydroxybenzaldehyde,while the main metabolites of D3R were gallic,syringic acid and 2,4,6-trihydroxybenzaldehyde.?3?The metabolism of C3G in vivo was evaluated in sprague-dawley?SD?rats.ACNs and metabolites were analyzed by HPLC-ESI-MS/MS.The results indicated that the pharmacokinetic parameters(C_max,T_max,MRT and AUC_0??)of C3G in vivo accord with one-compartment model.The pharmacokinetic trial of C3G in rats showed a rapid elimination in plasma,and a rapid absorption and metabolism in tissues.Native and methylated ACNs were identified in plasma,organ?brain,liver,kidney,stomach,intestine?,and excreta?urine and faeces?.The main metabolites of C3G in vivo were phenolic acids and its derivatives.The main metabolites in plasma and liver were PCA,PCA-glucoside,sulfate-PCA,hydroxybenzoic acid and vanillic acid;and the main metabolites in kidney and excreta?urine and faeces?were hippuric acid,ferulic acid and hydroxybenzoic acid;while in the intestine,a amount of PCA,vanillic acid,hydroxybenzoic acid and 2,4,6-trihydroxybenzaldehyde occurred as the main metabolites.?4?C3G and its main metabolite?PCA?were employed to elevate the effects on the chronic-Pb²⁺exposed SD rats,and found that C3G is the active ingredient with good ability to protect the nerve cells lesion.Behavioral experiments showed that L-C3G,M-C3G and H-C3G can reduce the escape latency of Morris maze test and active avoidance experiment of rats.The results of the studies on metal content in rats indicated that L-C3G,M-C3G and H-C3G can reduce the content of Pb²⁺in blood and tissues in rats,M-C3G and H-C3G can reduce the content of Pb²⁺in bones.The biochemical indexes of rats showed that L-C3G,M-C3G and H-C3G can increase the content of ACH,EEA,DA,NE,5-HT,NO and the activity of NOS,SOD and GSH-Px in brain;L-C3G,M-C3G and H-C3G can improve the activity of SOD and GSH-Px,and rdduce the level of MDA in liver;L-C3G and M-C3G can improve the content of transaminanes?ALT and AST?in the blood,improve the activity of SOD,and rdduce the level of MDA in kidney.The results indicated that C3G can reduce the accumulation of Pb²⁺in rats,improving the oxidative damageand the expression of the enzymes of learning and memory caused by Pb.?5?C3G was employed to intervene the mechanism of inhibition of cAMP-PKA-CREB signaling pathway in Pb²⁺-induced neurons.The results showed that C3G can promote the expression of excitatory amino acid receptors NMDA,accerelate extracellular Ca²⁺entry into cells through voltage-dependent calcium channels and NMDA receptor channels,activate CaMKII,PKC,and further activate nNOS oxidative activity,promoting nNOS oxidation toproduce NO.Ca²⁺influx led to the activation of calmodulin,and catalyzed the synthesis of cAMP through adenylyl cyclase,which in turn activated PKA and feeds back to the presynaptic membrane through diffusion,producing large amounts of glutamate.After the PKA and PKC enter the nucleus,the phosphorylation CREB?p-CREB?occurred and promoted the synthesis of new proteins related to learning and memory;at the same time,the second messenger cAMP activated PKA and accompaniedthe expression of the immediate early gene,which promoted the conversion of short-term memory into long-term memory and the formation of Long-term potentiation?LTP?,thereby enhancing learning and memory ability.The intervention mechanism showed that C3G could promote the expression of cognitive proteins and genes in the cAMP-PKA-CREB signaling pathways,and improve the ability of learning and memoryin Pb²⁺exposed rats.It could provide a scientific basis for the application of the mulberry anthocyanins,and nutrition intervention on nerve cell damage induced by Pb²⁺.