Exploring The Protective Effect And Mechanism Of Icariin In H₂O₂-Induced HT22 Oxidative Damage Based On NRF2

Objective:Based on the results of the previous network pharmacology experiments,we investigated the protective effects and mechanisms of Icariin（ICA）on H₂0₂-induced oxidative damage in HT22 cells,which simulates the oxidative stress state to which nerve cells are subjected in AD.Methods:（1）Preparation of HT22 oxidative damage model and screening of ICA concentration.The CCK-8 method was used to screen the appropriate H₂O₂ concentration to induce oxidative damage model of HT22 and the ICA concentration and duration of action.（2）Experimental grouping.Subsequent experiments were grouped according to the screened H₂O₂ and ICA concentrations,grouped as follows:Normal group（Control group）:HT22complete medium;Model group（H₂O₂group）:600μM H₂O₂;ICA low concentration group:600μM H₂O₂+5μM ICA;ICA medium concentration group;ICA high concentration group.（3）Determination of antioxidant capacity of ICA.Including the detection of oxidation-related indicators ROS,GSH,SOD,MDA,and the detection of apoptosis protein expression levels by Western blot.（4）Effects of ICA on the expression levels of related genes and proteins in NRF2 pathway.The m RNA levels of NRF2,HO-1,NQO1 and Keap1 were detected by q RT-PCR.The expression levels of NRF2,HO-1 and NQO1 proteins were detected by Western blot.（5）The expression of NRF2 in the cells was observed by immunofluorescence staining.（6）Finally,molecular docking was performed between ICA and Keap1/NRF2 complex to further explore the mechanism by which ICA activates NRF2 in the cytoplasm.Results:（1）Treatment with 600μM H₂O₂ for 24h was selected as the appropriate condition for modeling.Appropriate concentration of ICA could improve the cell survival rate of H₂0₂-induced oxidative damage model of HT22 cells（P<0.05）,and make the cell growth morphology tend to be normal.（2）ICA increased the content of antioxidant indicators GSH and SOD（P<0.05）and decreased the content of ROS and lipid oxidation index MDA（P<0.05）in the HT22 model of oxidative damage,and this effect of ICA showed some dose dependence.（3）The q RT-PCR results showed that ICA could differentially increase the m RNA levels of NRF2,HO-1 and NQO1（P<0.05）,but there was no significant effect on the m RNA level of Keap1（P>0.05）.Western blot showed that ICA increased the expression levels of NRF2,HO-1,NQO1 and Bcl-2（P<0.05）and decreased the expression of Bax（P<0.05）in the HT22 oxidative damage model,and this effect showed a certain degree of dose dependence.Immunofluorescence results showed that ICA caused a dose-dependent increase in the expression of NRF2 in the HT22 oxidative damage model.Under normal conditions,NRF2 binds to Keap1 mainly in the cytoplasm,whereas NRF2 expression and nuclear translocation were impaired under oxidative stress.The treatment of ICA alleviated this impairment and increased nuclear translocation of NRF2.Molecular docking results show that ICA binds well to the Keap1/NRF2 complex,and the binding site of ICA-Keap1/NRF2 is close to the binding site of the Keap1/NRF2 complex and interacts with each other.Conclusion:（1）ICA treatment enhanced the antioxidant capacity of cells,reduced the damage caused by oxidative stress,and inhibited the activation of apoptotic pathways caused by oxidative stress.And this antioxidant capacity of ICA showed a dose-dependent effect in this study.（2）In neuronal cells under oxidative stress,ICA could increase the nuclear translocation of NRF2 and the expression of NRF2 and its downstream genes HO-1 and NQO1 to enhance the antioxidant capacity of the cells.（3）ICA may compete with NRF2 for the binding sites with Keap1,leading to the release and activation of NRF2 from the Keap1/NRF2 complex in the cytoplasm,increasing its nuclear translocation to initiate the expression of its downstream genes.（4）In this study,the pathological changes of AD was linked as a whole through the role of oxidative stress as a"bridge"in AD under the guidance of holistic concept in Traditional Chinese Medicine（TCM）.This study showed that ICA could activate NRF2 to regulate oxidative stress and therefore could participate in various pathological pathways and progression stages of AD,which could affect the development of AD in a holistic manner.This study provided an experimental basis for kidney tonics method in the clinical treatment of AD in TCM,which also provided new ideas for the research on the mechanisms targeting AD.