Protective Effects Of Myricetin And Morin On Aβ₄₂/Al³⁺-Induced Nervous System Damage And Apoptosis In Rats

ObjectiveThe number of people with Alzheimer’s disease（AD）continues to increase,and the social and economic burden is becoming increasingly obvious,which has become a major disease and social problem seriously threatening the health of Chinese population.The pathogenesis of AD is not clear.The main pathological features are extracellular amyloid plaque,intracellular neurofibril tangles and neuronal death.The current treatment methods can not completely cure AD,which are accompanied by various degrees of adverse outcomes.According to the drug development model of multi-target strategy,natural small molecular compounds have great potential and unique advantages in the treatment of AD because of their low toxicity,diverse chemical structure and wide range of biological activities.Among them,flavonoids have been concerned by researchers because of their strong biological activity.Myricetin and morin belong to flavonol subclass of flavonoids.Their chemical structures of polyhydroxyl groups and multi-coordination sites are superior in many drug screening experiments and have the potential to become anti-AD drugs.In this study,according to the most recognized hypothesis of β-protein cascade,SD rats were treated with intracerebroventricular injection of Aβ₄₂ and intragastric administration of A13+to establish a multi-factor compound animal model of AD rats,and then the AD rats were treated with myricetin and morin.Through the multi-angle analysis of the levels of Aβ and Tau,element levels,apoptosis and inflammatory factors,in order to explore the protective effects of myricetin and morin on nervous system injury and apoptosis induced by Aβ₄₂/Al³⁺.MethodsNinety six SD rats of SPF grade were divided into experimental group（n=84）and negative control group（n=12）at random.The AD rat model was established by intracerebroventricular injection of Aβ₄₂（2 μg/μL,5 μL）and intragastric administration of aluminum trichloride hexahydrate solution（281.4 mg/kg/d for 4 weeks）,but in the negative control group,rats with PBS and normal saline for 4 weeks.After the animal model was established successfully,the experimental group falls into model group,and low-dose,middle-dose and high-dose myricetin group,and low-dose,middle-dose and high-dose morin group（n=12）.The low,middle and high intervention groups were treated with myricetin or morin with 50 mg/kg/day,100 mg/kg/day and 200 mg/kg/day,respectively.Rats in the negative control group and model group were given 0.5%carboxymethyl cellulose sodium of the same volume by intragastric administration every day.The administration time was 8 weeks.At the end of administration,two rats in each group were randomly selected to make brain slices to evaluate the changes of neuronal apoptosis（TUNEL staining）,amyloid plaque deposition（sulfur staining）and hippocampal morphology（HE staining）.After decapitation,the serum of the other rats was collected and the brain tissue and hippocampus were separated and stored at-80℃ for followup analysisThe content of Aβ and Tau protein,the expression of apoptosis protein Bcl-2,Bax,Caspase-3 and inflammatory factors TNF-α,caspase-1,IL-1β and IL-6 in rat hippocampal lysates were detected by westernblot,and the mRNA expression levels of TNF-α,Caspase-1,IL-1β,IL-6 and other inflammatory factors in rat hippocampus were detected by real-time fluorescence quantitative PCR.Determination of calcium,magnesium,copper,aluminum,iron and zinc in brain and serum by inductively coupled plasma atomic emission spectrometry.Results1.The effect of myricetin and morin on Aβ and Tau in hippocampus of ratsCompared to control group,the protein expression of Aβ,Tau and p-Tau in hippocampus of model group was significantly increased（p<0.05）.Compared to model group,the protein expression of Aβ in high dose myricetin group and middle and high dose morin groups decreased significantly（p<0.05）,and Tau in the middle and high dose myricetin groups and the middle and high dose morin groups decreased（p<0.05）,and p-Tau in the middle and high dose myricetin groups and the low,middle and high dose morin groups decreased（p<0.05）.2.The effect of myricetin and morin on the deposition of amyloid fiber plaques in the brain of ratsThe number of amyloid fiber plaques of model group was more than that in control group（p<0.05）.The number of plaques in the middle and high dose myricetin groups and the middle and high dose morin groups decreased significantly（p<0.05）.3.The effect of myricetin and morin on hippocampal morphology of ratsHippocampal neurons in the negative control group showed relatively uniform round or oval shape,relatively complete structure,neat arrangement and tight distribution,and no pathological changes were found.The distribution of hippocampal neurons in the model group was loose,arranged disorderly,and there were more cell vacuoles.After intervention,the hippocampal neurons in the middle-dose myricetin group,high-dose myricetin group and highdose morin group were more closely distributed,neatly arranged and here were less vacuoles than those in the model group.4.The effect of myricetin and morin on apoptosis of hippocampal neurons in ratsThe hippocampal cells of the model group showed obvious apoptosis,and the proportion of positive cells in the negative control group was more than that in control group（p<0.05）.The proportion of positive cells in myricetin low,middle and high dose groups and morin low,middle and high dose groups decreased significantly（p<0.05）.5.The effect of myricetin and morin on the expression of apoptosis-related proteins in the hippocampus of ratsThe expression of Bax and cleaved Caspase-3 protein in the hippocampus of the model group was more than that of the control group（p<0.05）.The expression of Bax protein in middle and high dose myricetin groups and high dose morin group decreased significantly（p<0.05）.The expression of cleaved Caspase-3 protein in middle and high dose myricetin groups and high dose morin group decreased significantly（p<0.05）.The ratio of Bcl-2/Bax in the model group was lower than that in control group（p<0.05）,while that in the high-dose myricetin group and morin high-dose group was significantly increased（p<0.05）.6.The effect of myricetin and morin on the levels of elements in brain tissue and serum of ratsIn brain tissue,the content of aluminum in model group and all intervention groups was much higher than that of the control group（p<0.05）,and the content of calcium of model group decreased（p<0.05）,and zinc of the model group decreased（p<0.05）,but there was no significant difference of aluminum,calcium and zinc between the intervention groups and the model group.Compared to control group,the content of iron of model group increased significantly（p<0.05）,while that of the middle-dose myricetin group and high-dose morin group decreased（p<0.05）.Compared to negative control group,the contents of calcium,iron,magnesium and zinc in the serum of the model group decreased significantly（p<0.05）,but there was no significant difference between the intervention groups and the model group.7.The effect of myricetin and morin on the expression of inflammatory factor-related mRNA and protein in the hippocampus of ratsCompared with the negative control group,the mRNA expression levels of TNF-α,IL-1β,IL-6 and Caspase-1 in the hippocampus of the model group were increased（p<0.05）.The mRNA expression of TNF-α in hippocampus of rats in high dose myricetin group and middle and high dose groups of morin was higher than that in control group（p<0.05）.The mRNA expression level of IL-1β in hippocampus of rats in middle and high dose of myricetin and middle and high dose of myricetin decreased significantly（p<0.05）.The expression of IL-6 mRNA in low,middle and high dose of myricetin groups and low,middle and high dose of morin groups decreased significantly（p<0.05）.The mRNA expression of Caspase-1 in myricetin low,middle and high dose groups and morin low,middle and high dose groups decreased significantly（p<0.05）.Compared with the negative control group,the protein expression levels of TNF-α,IL-1β,IL-6 and Caspase-1 in the hippocampus of the model group were significantly increased（p<0.05）.Compared with the model group,the protein expression of TNF-α in the hippocampus of rats in low,middle and high dose myricetin groups and middle and high dose morin groups decreased significantly（p<0.05）.The protein expression level of IL-1β in hippocampus of rats in middle and high dose groups of myricetin and morin decreased significantly（p<0.05）.The protein expression level of IL-6 in middle and high dose myricetin groups and middle and high dose groups of morin decreased significantly（p<0.05）,and the protein expression level of Caspase-1 in middle and high dose groups of myricetin and low,middle and high dose groups of morin decreased significantly（p<0.05）.ConclusionsMyricetin and morin alleviate neuroinflammation in the brain by down-regulating inflammatory cytokines;myricetin and morin could reduce the expression of Caspase family apoptosis-executing proteins of the Bcl-2 family pro-apoptotic proteins,and alleviate the progress of neuronal apoptosis;myricetin and morin could sequester iron to reduce the accumulation of iron in the rat brain;myricetin and morin could reduce the content of Aβ and Tau,which is directly related to the occurrence and development of AD,and inhibit the formation of pathological amyloid plaques,which further improve the neuronal morphology and reduce hippocampal neurons apoptosis.Myricetin and morin have protective effects on Aβ₄₂/Al³⁺induced nervous system damage.