Mechanism Study Of Myricetin Improving Cognitive Impairment In 3×Tg-AD Mice

Background:Alzheimer’s disease（AD）is a neurodegenerative disease,its main pathological features areβ-amyloid（Aβ）deposition and neuronal damage.Clinically,although there are drugs for the treatment of AD,they can’t improve significantly the neuropathy of patients.Myricetin is a natural flavonoid,the studies found that it has neuroprotective effects.It can inhibit neuronal damage in the hippocampal CA3 area of AD animal models and improve cognitive impairment.Objective:3×Tg-AD mice were used as the model of Alzheimer’s disease to evaluate the therapeutic effect of flavonoid（myricetin）on AD and explore the mechanism of myricetin that improved AD.Methods:（1）The study used eight months WT mice and 3×Tg-AD mice of the same age,and divided them into five groups:wild control group（WT-Con）,model group（3×Tg-AD）,myricetin administration group（3×Tg-AD+Myr 10 mg/kg,3×Tg-AD+Myr 20 mg/kg）,positive drug control group（3×Tg-AD+Donepezil 1 mg/kg）.The administration group mice administrated myricetin by intraperitoneal injection and the wild control group,model group and positive drug control group were injected intraperitoneally with the same solvent and positive drug.Dosing cycle is 21days.At the same time,BV2 cells were selected and Aβ_25-35 was used to induce BV2 cells to construct an in vitro AD model,and the molecular mechanism was verified by using the P38 MAPK signaling pathway agonist dehydrocorydaline（DHC）.Six experimental groups were set up:Control group（Con）:BV2 cells with solvent,control+administration group（Con+Myr）,in vitro AD model group(Aβ_25-35 10μmol/L),model+administration group(Aβ_25-35 10μmol/L+Myr 50μmol/L),model+agonist group(Aβ_25-35 10μmol/L+DHC 10μmol/L),model+agonist+administration group (Aβ_25-35 10μmol/L+DHC 10μmol/L+Myr 50μmol/L).（2）Water maze test was conducted after the drug administration to evaluate the spatial learning and memory ability of the mice.Nissl staining was used to observe the changes of hippocampal and cortical neurons in paraffin section.The Amyloid plaque in paraffin section was observed by Thioflavin S-staining.Enzyme-linked immunosorbent assay（ELISA）was used to detect levels of interleukin-1β（IL-1β）,tumor necrosis factor-α（TNF-α）,interleukin-6（IL-6）,interleukin-4（IL-4）and interleukin-10（IL-10）in the hippocampus and cortex in mice.Expression of Iba1,Arg1 and CD16 in cortex in mice detected by immunofluorescence staining.Network pharmacology,molecular docking technology and Cellular Thermal Shift Assay（CETSA）experiments were used to study the combination of Myricetin and P38.Mitochondrial membrane potential detection Kit was employed to examine the mitochondrial membrane potential of BV2cells.The expressions of p-P38,dynamin-related protein 1（DRP1）,mitochondrial fission protein 1（FIS1）,optic atrophy 1（OPA1）,mitofusin 2（MFN2）,nuclear respiratory factor 1（NRF1）,mitochondrial transcription factor A（TFAM）,NOD-like receptor thermal protein domain associated protein 3（NLRP3）,apoptosis-associated speck-like protein containing a CARD（ASC）,cysteinyl aspartate specific proteinase-1（caspase-1）,interleukin-18（IL-18）,Iba1,Arg1,CD16,postsynaptic density protein-95（PSD95）,recombinant synaptophysin（SYP）were detected in fresh brain tissues and cells by western blot.Results:（1）Pharmacodynamic study of myricetin in improving cognitive dysfunction in 3×Tg-AD mice.All groups showed a certain learning ability during the 5-day training in Morris water maze experiment.The incubation period of 3×Tg-AD mice to reach the platform increased significantly compared to WT group,and the latency of the mice in the administration group was shorter than 3×Tg-AD mice.When the test platform was hidden,3×Tg-AD mice exhibited an aimless search strategy,whereas myricetin-treated mice spent more time in the target quadrant and the distance in the target quadrant.The frequency of platform crossing of Myr 20 mg/kg group was similar to the positive drug donepezil group and the effect was close to that of WT group.The effect of Myr on AD pathology was further explored by observing the Aβplaque deposition and neuronal loss in the brains of mice in WT group,3×Tg-AD group,and 3×Tg-AD+Myr 20mg/kg group.The study findings indicated that Myr attenuated the burden of Aβplaques in the cortex and ameliorated the pathological deterioration of cerebral neurons in 3×Tg-AD mice.Meanwhile,the western-blot assay detected the expression of postsynaptic density protein（PSD95）and synaptophysin（SYP）,suggesting that Myr may promote synaptic repair in the hippocampus and cortex of 3×Tg-AD mice.（2）Myricetin targets and inhibits the activation of P38 MAPK pathway in the brain of 3×Tg-AD mice.The results of network pharmacology and molecular docking revealed that Myr could act on various targets to regulate MAPK signaling pathway.And the Myr was docked with P38 and produced binding energy of–7.4 kcal/mol,indicating the stability of Myr binding to P38.The results of CETSA exhibited that the denaturation temperature of P38 differed by 52–61℃with and without Myr,revealing that Myr could bind directly to P38.Furthermore,the Western blot results revealed that phosphorylated P38 levels in the cortex and hippocampus of 3×Tg-AD mice were significantly higher than those in the WT group.This finding indicated that P38 MAPK signaling pathway was activated in the mice model but inhibited by Myr.（3）Myricetin restores mitochondrial dynamic equilibrium and promotes mitochondrial biogenesis in3×Tg-AD mice brain tissue.Western blot results showed that the expressions of FIS1 and DRP1 were significantly higher in the cortex and hippocampus of 3×Tg-AD mice than those of WT mice.On the contrary,the expressions of OPA1 and MFN2 were reduced,indicating that the balance between mitochondrial fusion and fission is compromised in this model.In addition,the protein expression levels of NRF1 and TFAM decreased distinctly in the brain tissue of 3×Tg-AD mice,implying the abnormality in the mitochondrial biogenesis.These results were reversed post-Myr treatment,revealing the Myr potential in restoring the dynamic balance between mitochondrial fission and fusion in the brain tissue of 3×Tg-AD mice and promoting mitochondrial biogenesis.（4）Myricetin inhibits NLRP3 inflammasome activation.Western blot results revealed that NLRP3,ASC,Caspase-1,and IL-18 expressions in the brain tissue of 3×Tg-AD mice were significantly higher than those of WT mice and this means that NLRP3 inflammasome was activated in the brain tissue of 3×Tg-AD mice.In contrast,Myr decreased the expression of NLRP3,ASC,caspase-1,and IL-18,implying that Myr inhibited NLRP3 inflammasome activation.（5）Myricetin inhibits microglia hyperactivation and promotes the transformation of microglia from M1type to M2 type.Immunofluorescence staining and western blot found that the microglia marker（Iba1）and M1 marker（CD16）expressions increased significantly,whereas the expression of M2 marker（Arg1）decreased in the cortex of 3×Tg-AD mice.The situation was reversed by Myr treatment,indicating that this flavonoid could significantly inhibit microglia hyperactivation in 3×Tg-AD mice and promote the transformation of microglia from M1 type to M2 type.（6）Myricetin improves neuroinflammation in 3×Tg-AD mice.ELISA was used to detect pro-inflammatory factors（IL-1β,TNF-α,and IL-6）and anti-inflammatory factors（IL-4 and IL-10）in the hippocampus and cortex to evaluate the effect of Myr on neuroinflammation in 3×Tg-AD mice.The results exhibited that IL-1β,TNF-α,and IL-6 expressions decreased,while IL-4 and IL-10 significantly increased in the 3×Tg-AD+Myr 20 mg/kg treatment group.Consequently,Myr could effectively inhibit neuroinflammation in 3×Tg-AD mice.（7）Myricetin inhibits the activation of P38 MAPK pathway in AD in vitro model.Aβ_25-35（10μmol/L）was used to induce BV2 cells for 24 h to build an in vitro AD model,while DHC（10μmol/L）was used to verify whether the regulation of Myr（50μmol/L）on mitochondria-NLRP3 inflammasome-microglia channel was related to the activation of P38 MAPK signaling pathway after 48 h.Western blot results revealed that Myr effectively inhibits P38 hyperphosphorylation induced by Aβ_25-35,while DHC induction further aggravated the P38 phosphorylation and promoted the activation of P38 MAPK signaling pathway.（8）Myricetin regulates mitochondria-NLRP3 inflammasome-microglia channel based on targeting P38MAPK signaling pathway.Western blot and JC-1 techniques were utilized in examining Myr functions on mitochondrial dynamics,biogenesis,and mitochondrial membrane potential via AD in vitro model.It was found that Myr could substantially restore the mitochondrial dynamics imbalance induced by Aβ_25-35,promote mitochondrial biogenesis,and maintain mitochondrial membrane potential by inhibiting the activation of P38 MAPK signaling pathway.What’s more,western blot findings illustrated that Myr could mitochondrial dysfunction induced by Aβ_25-35 by inhibiting the activation of P38 MAPK signaling pathway,thereby inhibiting the activation of the NLRP3 inflammasome.Western blot results also found that Myr could inhibit mitochondrial dysfunction induced by Aβ_25-35 by inhibiting the activation of P38 MAPK signaling pathway,thereby inhibiting the activation of the NLRP3 inflammasome,finally,the hyperactivation of microglia was inhibited and the transition from M1 to M2 type was promoted.Conclusions:Myricetin can improve the learning and memory ability of 3×Tg-AD mice,and alleviate the pathological changes of amyloid protein,neuron and synaptic damage in brain tissue of model mice.This mechanism may be Myr inhibited production of neuroinflammation by regulating the mitochondria-NLRP3inflammasome-microglia channel via targeting P38 MAPK signaling pathway.As a result,our findings support further development of Myr as a potential drug candidate for the treatment of AD.