Effects Of Buzhong Yiqi Decoction On H₂O₂-induced Oxidative Damage In PC12 Cells And Its Mechanism

Objective:The brain is the "house of spirit",which dominates the life activities of the human body and is the hinge of the activities of spirit,consciousness and thinking.Qi and blood harmony,Qingqi upward nutrition brain,brain can play a normal physiological function.TCM clinical findings show that patients with qi deficiency syndrome have qi and blood weakness to develop the brain marrow and the loss of myeloid sea,which are prone to emotional,memory and cognitive dysfunction.As a representative prescription of qi replenishing agent.Buzhong Yiqi Decoction can improve the above symptoms by replenishing qi and raising Yang and invigorating the spleen.At present,there are few studies on the mechanism and molecular targets of Buzhong Yiqi Decoction in improving the symptoms related to the nervous system,and further research is needed.Modern research believes that mitochondria are the body's"energy factories",and the main source of energy for the brain.In our previous animal experiments,we found that rats with Qi-deficiency syndrome were accompanied by obvious symptoms of cognitive dysfunction,aggravated oxidative damage of neurons and abnormal changes in mitochondrial morphology and function.Buzhong Yiqi Decoction improves mitochondrial oxidative phosphorylation,reduces the generation of oxygen free radicals,and alleviates oxidative damage of neurons through the effect of replenishing qi,which may be one of the molecular mechanisms of Buzhong Yiqi Decoction in improving brain cognitive dysfunction.Therefore,the oxidative damage model of PC 12 cells was prepared in this study.By observing the effects of Buzhong Yiqi Decoction on mitochondrial function,morphology and related antioxidant pathways,the molecular mechanism of Buzhong Yiqi Decoction in improving cognitive dysfunction was further explored from the cellular level.In order to reveal the modern biological essence of "replenishing qi" in traditional Chinese medicine,we observe the effect of the lifting characteristic of the medicine in buzhong yiqi decoction on neurons by means of disconnecting the prescription.Methods:1.PC 12 cell injury model was prepared by H2O2.The concentration groups of 100,200,400 and 800 M H2O2 were set.Cell activity was detected by CCK-8 and mitochondrial morphology was observed to select the appropriate concentration of H2O2 to prepare the oxidative damage model of PC12 cells.2.Observe the effects of Buzhong Yiqi Decoction and its disjoint formula on the morphology and function of mitochondria in the oxidative damage model of PC12 cells.The normal group,model group,Danakan group,Huaifang 1 group,Huaifang 2 group and Buzhong Yiqi Decoction group were set.CCK-8 assay was used to detect the cell activity,Mito-Red fluorescent probe was used to observe the changes of mitochondrial morphology and network structure,JC-1 assay was used to detect the changes of mitochondrial membrane potential,and colorimetric assay was used to detect the activity of I-complex of mitochondrial respiratory chain and the content of ATP in each group.3.To observe the effects of Buzhong Yiqi Decoction and its decoction on SIRT3/MnSOD/OGG1 pathway and mtDNA oxidative damage in PC 12 cell model.The cells were grouped as above.The protein levels of SIRT3,MnSOD and OG1 in each group were detected by Westen Blot,and the content of 8-hydroxydeoxyguanosine(8-OHdG)was detected by ELISA,The ROS level was detected by fluorescence probe labeling.Results:1.PC12 oxidative damage model induced by H2O2 was prepared:PC12 cells were treated with H2O2 at a concentration of 100 M for 3 hours,and cell activity decreased,mitochondrial morphology changed,network structure was broken,and oxidative damage was caused to mitochondria.Therefore,this concentration was determined to be the best concentration for the preparation of the model.2.Buzhong Yiqi Decoction and its decoction can effectively improve the abnormality of mitochondrial morphology and function in the oxidative damage model of PC 12:First,16mg/mL of Buzhong Yiqi Decoction was selected as the best concentration for administration.CCK-8 results showed that Buzhong Yiqi Decoction and its extract significantly improved the mitochondrial activity of PC 12 cells with oxidative injury.Fluorescence microscope observation showed that the number of mitochondria in PC12 cells was less and the network structure was loose and dotted.The abundance of mitochondria in Buzhong Yiqi Decoction and its two decoction groups was increased,while the number of dotted mitochondria and tubular mitochondria were significantly reduced,suggesting that the structure of mitochondria network was improved.Quantitative analysis of average branch number and average branch length of mitochondrial network in each group showed that the group of Buzhong Yiqi Decoction was superior to the two groups(P<0.001).Flow cytometry was used to detect the fluorescence intensity of JC-1 polymer and JC-1 monomer,which indicated that the mitochondrial membrane potential of each treatment group was increased compared with that of the model group.Mitochondrial respiratory chain complex I-activity and ATP content test results showed that compared with the normal group,the cellular respiratory chain complex proliferation activity and ATP content in model group were significantly decreased(P<0.001).Compared with model group,the activity of mitochondrial respiratory chain complex I-component and the content of ATP in all administration groups were increased(P<0.05,P<0.01,P<0.001),and the contents of rhizoma and alum of respiratory chain complex in Buzhong Yiqi Decoction group were higher than those in the undecoction group 2(P<0.05).There were no significant differences in the activities of the four respiratory chain complex and the content of ATP between the two groups.3.Buzhong Yiqi Decoction and its decoction can regulate SIRT3/MnSOD/OGG1 pathway and reduce oxidative damage of mtDNA:Westen Blot results showed that compared with the normal group,the protein expression levels of SIRT3 and OGG1 in model group were significantly decreased,and the acetylation level of MnSOD was significantly increased(P<0.001).Compared with model group,the expression of SIRT3 and OGGI of Buzhong Yiqi Decoction and its decoction were significantly increased,and the acetylation level of MnSOD was decreased(P<0.01,P<0.001).Compared with the three TCM groups,the protein content of SIRT3 in Buzhong Yiqi Decoction group was higher than that in the decoction group 2(P<0.05).In terms of reducing MnSOD acetylation and upregulating OG1 expression,it was superior to the two decoction groups.8-OHdG and ROS content test results showed:The contents of 8-OHdG and ROS in the model group were significantly increased(P<0.001).Compared with the model group,the contents of ROS and mitochondrial 8-OHdG in each treatment group were decreased(P<0.001).The effect of Buzhong Yiqi Decoction group was better than that in the disjoint formula 1 and disjoint formula 2 groups(P<0.001),and there was no statistical difference between the two groups.Conclusion:1.fill in yiqi decoction can effectively improve the mitochondrial morphology and function of PC 12 cell damage induced by H2O2,tip resistance to oxidative damage and protection of mitochondrial function,may be one of the internal mechanism of the party play a role of nerve protection,from the cellular level to "air" of traditional Chinese medicine treatment to improve the deficiency of card provides experimental basis of cognition;At the same time,it also provides further experimental evidence for the relationship between "qi" in Chinese medicine and mitochondria.2.Buzhong Yiqi Decoction can enhance antioxidant function,reduce cellular ROS content and reduce oxidative damage of mtDNA by regulating SIRT3/MnSOD/OGG1 pathway,which may be one of the molecular targets of this compound to improve oxidative damage of mitochondria.3.The whole prescription of Buzhong Yiqi Decoction was superior to the two unpacked prescriptions in terms of improving the structure of mitochondrial network,enhancing the function of respiratory chain and regulating the signal pathway of antioxidant damage,indicating that the combination of traditional Chinese medicine prescriptions has the therapeutic advantage of integrating regulation.