Study On The Protective Effect And Mechanism Of Periplaneta Americana Extract On H₂O₂ And Glu Induced PC12 Cell Injury

Objective:By using H₂O₂induced rat adrenal pheochromocytoma cells（PC12）to establish oxidative damage model of nerve cells in vitro,and investigated the protective effect of Periplaneta americana extract（PAS840）on the cells and the effect of the Nrf2/HO-1pathway.By using Glu induced PC12 cells to establish excitatory neurotoxicity model in vitro,and investigated the protective effect of PAS840 and its impact on the expression of NMDAR1.This discussion provided a theoretical framework and data support for the use of Periplaneta americana extract in the study of neurodegenerative diseases.Methods:1.The CCK-8 assay was used to detect the effect of PAS840 on the survival rate of PC12 cells,screen the safe dose range of PAS840,and determine the concentration for subsequent experiments.2.The H₂O₂-induced PC12 cells for 12 hours to established the oxidative damage model,and Glu-induced PC12 cells for 24 hours to established the excitatory neurotoxicity model.3.The biochemical assay was used to detect the effect of PAS840 on the levels of SOD,GSH-Px,LDH,GSH,and MDA in the two model cells;DCFH-DA fluorescent probe was used to detect the effect of PAS840 on the ROS fluorescence intensity in the oxidative damage model cells;and the calcium content was detected by colorimetric assay to evaluate PAS840’s impact on the Ca²⁺level in the excitatory neurotoxicity model cells.4.Flow cytometry was used to detect the effect of PAS840 on apoptosis in the two model cells and he JC-1 fluorescence labeling method was used to detect the effect of PAS840 on the MMP in the two model cells.5.RT-PCR was used to detect the effect of PAS840 on the m RNA expression levels of Nrf2/HO-1 pathway factors（Nrf2,Keap1,HO-1,and NQO1）,apoptosis factors（Bcl-2,Bax,and Caspase-3）,inflammatory factors（TNF-α,IL-1β,and IL-6）,and CAT in the oxidative damage model cells;and to detect the effect of PAS840 on the m RNA expression levels of NMDAR1,Cytc,Bcl-2,Bax,Caspase-3,TNF-α,and IL-6 in the excitatory neurotoxicity model cells.6.ELISA was used to detect the effect of PAS840 on the expression levels of TNF-α,IL-1β,and IL-6 in the culture medium of the two model cells.7.Western blot was used to detect the effect of PAS840 on the protein expression levels of Nrf2,HO-1,Bcl-2,Bax,and Caspase-3 in the oxidative damage model cells;and to detect the effect of PAS840 on the protein expression levels of NMDAR1,Cytc,Bcl-2,Bax,and Caspase-3 in the excitatory neurotoxicity model cells.Results:1.The logarithmic growth period of PC12 cells is 24 h-72 h.According to cell survival rate,PAS840 concentrations of 20,50 and 125μg·m L^-1were selected as low（PAS840-L）,medium（PAS840-M）and high（PAS840-H）dose for subsequent experiments.2.The protective effect of PAS840 on oxidative damage model:After 12 h of 250mmol·L^-1H₂O₂induction,PC12 cells synapses decreased and crumpled,and the survival rate decreased significantly,ROS fluorescence intensity increased（P<0.05）,and occurred oxidative damage.In contrast,PAS840 increased the survival rate of damaged cells,reduced ROS fluorescence intensity,upregulated the levels of SOD,GSH-Px,GSH,and CAT,and decreased the levels of LDH and MDA.Moreover,the PAS840-H group significantly improved cell’s MMP,downregulated the expression of inflammatory factors TNF-α,IL-1β,and IL-6 in damaged cells and culture medium,and reduced the apoptotic rate of cells（P<0.01）.3.The protective mechanism of PAS840 on the oxidative damage model:PAS840decreased the expression of Nrf2 and Keap1,regulated the expression of downstream NQO1 and HO-1,further downregulated the expression of Bax and Caspase-3,and upregulate the expression of Bcl-2.4.The protective effect of PAS840 on excitatory neurotoxicity model:After treated with 30 mmol·L^-1Glu for 24 h,the survival rate of PC12 cells was significantly decreased and Ca²⁺level was significantly increased（P<0.01）.Compared with the model group,PAS840 reduced the levels of Ca²⁺,LDH,MDA and cell apoptosis in damaged cells,increased the level of cell survival rate and the levels of SOD,GSH-Px and GSH.At the same time,PAS840 can reduced the expression of TNF-α,IL-1β,and IL-6 in damaged cells and culture medium.5.The protective mechanism of PAS840 on excitatory neurotoxicity model:PAS840 inhibited the activity of NMDAR1 in damaged cells,improve the MMP of cells,down-regulated the expression of Cytc,Bax,and Caspase-3,and up-regulated the expression of Bcl-2 in damaged cells.Conclusions:1.PAS840 can regulate Nrf2/HO-1 signaling pathway factors to reduce oxidative damage degree of cells,improve antioxidant capacity of cells,up-regulate MMP of damaged cells,reduce inflammation,and then inhibit H₂O₂-induced apoptosis of PC12cells.2.PAS840 can inhibit the expression of NMDAR1 in PC12 cells induced by Glu,reduce intracellular Ca²⁺accumulation,protect mitochondrial morphology,reduce the release of Cytc,reduce inflammation,improve antioxidant levels,and inhibit cell apoptosis.