| Objective:Traditional Chinese medicine holds the view that the spleen in storing idea and in charge of thoughts,and the spleen’s functions of transporting and transforming of nutrients from foodstuff,and sending up essential substance make the brain nourished,thus exerting the function of thinking and memory.If the spleen-qi is deficient,the lack of qi and blood will lead to mind dystrophy,manifesting as symptoms such as inattention and decreased learning and memory.Clinically,it has been proved that the treatment method of strengthening spleen,benefiting qi and raising yang by traditional Chinese medicine can improve the above symptoms.However,the pathologic nature of cognitive decline caused by spleen-qi deficiency is still unclear;the molecular target of Buzhong Yiqi Decoction to improve cognitive symptoms is still lacking in-depth understanding.Modern medical research shows that the hippocampus is an important part of integrating visceral function,emotion,thinking,learning and memory.Insufficient energy will cause its dysfunction,and mitochondria as an important organelle for providing energy for life activities has great significance for the physiological function of the hippocampus.Deficiency of the spleen qi and dystrophy of the brain can lead to decreased aerobic metabolism of hippocampal nerve cells and increased free radicals,causing oxidative damage to mitochondria,affecting oxidative phosphorylation of it,and ultimately causing abnormal hippocampal function.This may be one of the molecular mechanisms of cognitive decline in patients with spleen-qi deficiency.Therefore,in this study,the model rats with spleen-qi deficiency syndrome were taken as the research object,and Buzhong Yiqi Decoction,a representative prescription for spleen-qi deficiency,was used as an intervention method.To explore the pathological essence of cognitive decline caused by spleen qi deficiency syndrome from the perspective of oxidative damage of hippocampal neurons,and to reveal the molecular mechanism of improving cognitive ability by Buzhong Yiqi Decoction.Methods:1.First,establish a rat model of spleen-qi deficiency syndrome by subcutaneous injection of reserpine.2.Observe the effect of Buzhong Yiqi Decoction on learning and memory ability of rats with spleen-qi deficiency:SD rats were randomly divided into 6 groups:namely normal group,model group,Danakan group(positive drug),decomposed recipes 1 group,decomposed recipes 2 groups and the Buzhong Yiqi Decoction.Use Quantified Score Table for Symptoms of Animal Model of Spleen-Qi Deficiency and serum D-xylose content were used to evaluate the symptoms of spleen-qi deficiency in each group of rats,and the open field experiment,Morris water maze experiment,shuttle experiment were used to test the autonomous activity and learning and memory ability.3.Observe the effect of Buzhong Yiqi Decoction on brain hippocampus mitochondrial structure and function of rats with spleen-qi deficiency.Animals were grouped and treated as above.The morphological structure of the hippocampus and the ultrastructure of mitochondria in neurons of each group were observed by HE staining and transmission electron microscopy.Use biochemical detection kit to detect the activity of mitochondrial respiratory chain enzyme complex Ⅰ-Ⅳ.Detection of ATP content in hippocampus by luminescence method to judge the change of mitochondrial function.4.Observe the effect of Buzhong Yiqi Decoction on SIRT3/MnSOD/OGG1 pathway in hippocampus of spleen-qi deficiency rats:Animals were grouped and treated as above.Extract mitochondria in rat hippocampus and use western blot to observe the level of SIRT3/SOD2/OGG1,furthermore,to assess the extent of oxidative damage to mitochondrial DNA in the hippocampus by detecting the content of 8-OH-dG.To find out whether Buzhong Yiqi Decoction and its decomposed recipes can act on the SIRT3/SOD2/OGG1 pathway to improved mitochondrial oxidative damage.Results:1.Buzhong Yiqi Decoction can significantly improve the spleen-qi deficiency symptoms of model rats and improve the learning and memory ability.According to the results of quantified score table,compared with normal group,model group weight gain slowed significantly(P<0.01),total score and individual scores were higher(P<0.05,P<0.01 P<0.001).Serum D-xylose were decreased(P<0.001).Compared with model group,medication group rats weight increased significantly,total score and individual scores decreased obviously(P<0.05,P<0.01,P<0.001),serum D-xylose content were increased(P<0.05,P<0.001).Buzhong Yiqi Decoction group has the most obvious effect(P<0.05,P<0.01,P<0.001),the effect of decomposed recipes 1 group was better than decomposed recipes 2 group.The open-field experiment,Morris water maze and shuttle experiments showed that the autonomous activity levels,space memory ability and active and passive learning abilities of rats in the model group decreased significantly compared with the normal group(P<0.05,P<0.01,P<0.001).After treatment,all the indexes were significantly improved compared with the model group(P<0.05,P<0.01,P<0.001).The open field experiment indexes of the Buzhong Yiqi Decoction group were better than Danakan group and the decomposed recipes 2 group(P<0.05,P<0.001),and the activity distance and average speed were better than those of the decomposed recipes 1 group(P<0.05,P<0.001).In the water maze experiment,many indexes of Buzhong Yiqi Decoction group were better than that of two decomposed recipes group(P<0.05,P<0.01,P<0.001).In the shuttle experiment,the Buzhong Yiqi Decoction group showed the active escape behavior,and the other indexes were also improved compared with the model group.2.Buzhong Yiqi Decoction can significantly improve the morphological abnormalities of hippocampus in rats with spleen deficiency syndrome:HE staining results showed that the hippocampal pyramidal cells of the rats were loosely arranged,the gaps were obvious,the cytoplasmic vacuolization,and the nucleus was obviously pyknosis.The hippocampal pyramidal cells in the Buzhong Yiqi Decoction group and the two decomposed recipes groups were improved compared with the model group,with increased cell levels and reduced cytoplasmic vacuolization.The effect of Buzhong Yiqi Decoction group is most obvious.3.Buzhong Yiqi Decoction can effectively improve the abnormalities of hippocampus mitochondria structural and functional in spleen-qi deficiency rats.Electron microscopy results showed that the morphology of mitochondria in the model group’s hippocampus had obvious abnormalities,serious vacuolation and swelling were seen,and there were mitochondria cristae fragmentation and rupture of mitochondrial membrane.After treatment,the mitochondria of each group have improved,the cristae fragmentation and vacuolation have decreased,the number of mitochondria has increased.Among them,the effect of Buzhong Yiqi Decoction group is best,the second is decomposed recipes 1 group,decomposed recipes 2 group.The detection results of the four mitochondrial complexes showed that the activity of mitochondrial complexes Ⅰ-Ⅳ in the model group decreased,which was significantly different from that in the normal group(P<0.05,P<0.001).Compared with the model group,the activity of the four mitochondrial complexes in each treatment group were increased,of which the activity of the four mitochondrial complexes in the Buzhong Yiqi Decoction group was significantly increased(P<0.05,P<0.01,P<0.001).Comparing the model group,only the increase of compound IV in the decomposed recipes 1 group was statistically significant(P<0.01).The comparison of the three traditional Chinese medicine groups showed that the four compounds’activity in the Buzhong Yiqi Decoction group were more increased than the decomposed recipes 2 group,no significant difference existed between the two groups.no significant difference existed between the two groups compatibility.Both were significantly increased(P<0.05,P<0.01,P<0.001),but there was no significant difference between the two split groups,showing the advantage of compatibility.After modeling,the mitochondrial ATP content of each model group decreased,which was significant compared with the normal group(P<005);compared with the model group,the mitochondrial ATP content of each treatment group increased(P<0.01,P<0.001),But there is no difference between the decomposed recipes group and the whole group.4.Buzhong Yiqi Decoction can reduce the oxidative damage of mtDNA by regulating the SIRT3/MnSOD/OGG1 pathway.Western Blot results showed that compared with the normal group,the SIRT3 protein expression of the model group was significantly decreased(P<0.05).Compared with the model group,the content of SIRT3 protein in each treatment group was significantly increased(P<0.01,P<0.001).Suggesting that each treatment group had varying degrees of increasing the content of SIRT3 protein in mitochondria,and the effects from high to low were in the order of Buzhong Yiqi Decoction group>decomposed recipes 1 group>decomposed recipes 2 group>Danakan group.About the acetylation of MnSOD protein shows that under normal circumstances,the degree of acetylation of MnSOD protein is very low,but after modeling,the acetylated MnSOD protein content increased significantly(P<0.001).That’s means Buzhong Yiqi Decoction can effectively reduce the level of MnSOD protein acetylation,and this effect is better than that of Danakan and the decomposed recipes groups(P<0.05);Compared with the normal group,the OGG1 protein expression in the model group was slightly reduced(P<0.01).Compared with the model group,the OGG1 protein content in each treatment group was significantly increased(P<0.01,P<0.001),It is suggested that Buzhong Yiqi Decoction and its decomposed recipes have effects on increasing the OGG1 protein content in mitochondria.The effect of Buzhong Yiqi Decoction group is better than that of decomposed recipes 2 group(P<0.01),however,there was no significant difference between Buzhong Yiqi Decoction and decomposed recipes 1 group.8-OH-dG content measurement results showed that:after modeling,the 8-OH-dG content in the mtDNA of rats in each experimental group was significantly increased(P<0.001).Compared with the model group,The content of 8-OH-dG of Buzhong Yiqi Decoction group and decomposed recipes 1 group decreased significantly(P<0.05,P<0.01),the remaining two groups were not statistically significant compared to the model group.The content of 8-OH-dG in decomposed recipes 1 group was lower than that of Buzhong Yiqi Decoction group and had significant difference(P<0.01).Conclusion:1.The rat model of spleen-qi deficiency caused by reserpine has obvious cognitive decline,mechanism research shows that the morphology of the hippocampus of the brain changes,the ultrastructure and function of mitochondria are abnormal,and mitochondrial oxidative stress injury occurs.Therefore,oxidative damage to the hippocampal mitochondria in the state of spleen deficiency leads to abnormal morphology and function of the mitochondria and hippocampus,which may be the internal pathological mechanism of the decline of learning and memory ability in spleen deficiency syndrome.2.Buzhong Yiqi Decoction can increase the expression of deacetylated protein SIRT3,reduce the degree of acetylation of MnSOD,and increase the expression of OGG1,thereby improving the state of mitochondrial oxidative damage.This may be one of the internal mechanisms of the prescription to improve the cognitive ability of patients with spleen deficiency syndrome.3.The study on the decomposed recipes of Buzhong Yiqi Decoction found that in improving the cognitive ability of the rats,the decomposed recipes 1 group is better than the decomposed recipes 2 group.The results of brain hippocampal mitochondrial function protection are consistent;suggesting a reasonable combination of Yiqi Shengyang herbs is of great significance in the treatment of learning and memory decline caused by ’Spleen fails in sending up essential substance’. |
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