| ObjectiveAlzheimer’ s disease(AD),commonly known as senile dementia,is an age-related degenerative central nervous system degenerative characterized by progressive memory and cognitive decline.disease.Recent studies have found that phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT),phosphatidylinositol 3-kinase/protein kinase B(PI3K/AKT)insulin and insulin-like nerve growth factor under oxidative stress The disorder of(insulin-like growth factor-1,IGF-1)plays an important regulatory role in the development of neurodegenerative diseases.Therefore,regulation of this signaling pathway is one of the ways to mitigate the progression of AD.Ferulic acid(FA)is a phenolic acid component found in various plants Angelica,Equisetum,Rhizoma Ligustium and Cimicifugae.It has good antioxidant activity and protects rat hippocampal neurons.However,the mechanism which ferulic acid exerts the above effects remains unclear.Therefore,at the cellular level,we evaluated the protective effect of ferulic acid on H2O2 mimicking oxidative stress pathology PC12,and evaluated whether its neuroprotective effect is related to the regulation of IGF-1/IGF-1R/PI3K/AKT,and provide experimental basis for the treatment of AD with ferulic acid.Methods1.Protective effect of ferulic acid on H2O2-induced oxidative damage in PC12 cells①Determination of oxidative damage dose and time Logarithmic growth phase cells were taken and inoculated in 96-well plates.100 μL of the system.After 24 hous of cell culture.The cells were stimulated by adding H2O2 solution at a final concentration of 0,100,150,200,250,300,350,400,450 μmol/L,and cultured at 37℃ in a 5%CO2 incubator for 6 h,12 h,and 24 h.The concentration of H2O2 at the inhibition rate of 30%-40%was determined as the optimal concentration of H2O2 induced PC12 cell injury,and the oxidative damage model was established at this concentration.②Determination of safe concentration of ferulic acid Logarithmic growth phase cells were seeded in 96-well plates.100μL system,after 24h cell adherent growth,the old medium was discarded,and the final concentration was(1,2.5,5,10,20,50,100,500 μmol/L)of FA solution,after 24 hours of culture,The safe concentration of ferulic acid was determined by MTT method.③Effect of ferulic acid on H2O2 injury in PC12 cells PC12 cells were stimulated with H2O2 with a cell inhibition rate of 30%to 40%,and the effect of ferulic acid at a safe concentration on the morphology and proliferation of PC12 cells was evaluated.The effect of FA on the proliferation of PC12 cells injured by H2O2 was detected by MTT assay.The number of cells and their morphological changes were observed under an inverted microscope.2.Mechanism of ferulic acid on H2O2-induced oxidative damage in PC12 cells①Experimental grouping divided into normal group,model group,low,medium and high doses of ferulic acid.Except the normal group,each group was stimulated with 350 mol/L H2O2,and the low,medium and high doses of ferulic acid were treated with ferulic acid at concentrations of 1,10 and 100 μmol/L.②Effect of ferulic acid on oxidative stress After co-cultivation of H2O2 and ferulic acid for 36 hours,the cell culture supernatant and cells were collected,and the levels of LDH and MDA in the supernatant and the SOD activity in the cells were measured.③Flow cytometry was used to detect the apoptosis rate of PC12 cells,and whether ferulic acid had protective effect on PC12 cells damaged by H2O2,.④RT-qPCR quantitative detection of FA on H2O2-induced IGF-1 mRNA expression in pc12 cells.⑤Western-blot was used to detect the expression of IGF-1,IGF-1R,PI3K,AKT and p-AKT protein in PC12 cells.Results1.The concentration of 350 μmol/L H2O2 can cause the cell viability of PC12 cells to decrease by 30%to 40%,so this concentration is used as the stimulation concentration for subsequent experiments.2.FA has a high safe concentration range and does not exhibit significant cytotoxicity at a concentration of 500 μ mol/L.3.FA has protective effect on PC12 cells induced by H202-induced oxidative damage Compared with the normal control group,the cell survival rate of the model group was significantly decreased(P<0.01),the number of cells was decreased,and the cell morphology was shrunk or rounded.After the administration of FA,the number and morphology of the cells in each administration group were compared.The group significantly increased and improved,and the cell survival rate was significantly increased compared with the model group under high dose(P<0.01).and the LDH level in the supernatant is significantly reduced.4.FA relieves oxidative stress caused by H2O2 Compared with normal control group,MDA content in model group is significantly increased(P<0.01),the activity of SOD enzyme was significantly decreased(P<0.05).Compared with the model group,the administration group significantly reduced the MDA content(the final concentration was 100 μmol/L,P<0.05)and significantly increased SOD.Enzyme activity(P<0.01).5.Effect of FA on H2O2-induced apoptosis of PC12 cells The results showed that compared with the normal control group,PC12 cells showed obvious apoptosis under H2O2 stimulation(P<0.01).After ferulic acid intervention,the apoptosis rate of PC12 cells decreased significantly,indicating that ferulic acid has inhibitory cells.The difference in the role of apoptosis was statistically significant(P<0.01).6.Effect of FA on IGF signaling pathway The results of RT-qPCR showed that the IGF-1 mRNA of PC12 cells in the model group was significantly lower than that of the normal control group(P<0.01),and the IGF-1 mRNA was significantly increased after FA treatment(P<0.01).Western-blot results showed that compared with the model group,ferulic acid could up-regulate the protein expression level of IGF-1/IGF1R/PI3K/AKT/pAKT in PC12 cells,and the protein expression trend was the same.It is suggested that the neuroprotective mechanism of ferulic acid may be related to the regulation of insulin-mediated proteins in the classical islet signaling pathway.Conclusions1.FA has a good protective effect on H2O2-induced PC12 cells in vitro,which can promote the proliferation of PC12 cells damaged by H2O2,which can protect the structural integrity of cell membrane,reduce the morphological changes of cells caused by external stimuli,and reduce cells.The leakage rate of LDH and the content of MDA in the supernatant also enhance the activity of SOD enzyme in the cells,enhance the antioxidant capacity of the cells,and inhibit the oxidative damage of the cells.2.FA has a neuroprotective effect by reducing apoptosis of PC12 cells due to oxidative stress.3.FA can significantly up-regulate the expression levels of IGF-1 mRNA and islet signaling pathway-related proteins IGF-1,IGF-1R,PI3K,AKT and p-AKT,and reveal that FA can improve islet signal transduction and regulate energy metabolism.Thereby playing a neuroprotective role. |
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