Gastrodin Protects Glutamate-induced Ferroptosis In HT-22 Cells Through Nrf2/HO-1signaling Pathway

Neurodegenerative diseases?ND?are complex nervous system diseases characterized by specific neurodegenerative lesions or massive neuronal loss.These include Alzheimer's disease?AD?,Parkinson's disease?PD?,Huntington's disease?HD?and amyotrophic lateral sclerosis?ALS?.Gastrodin is extracted from the dried root of Gastrodia elata,an orchid plant,and has strong antioxidant activity in ND.Excessive concentration of glutamate in intercellular space can cause neuronal toxicity,leading to neuronal degeneration,aging and death.This oxidative toxicity of glutamate is closely related to the occurrence and development of various ND,and is one of the important mechanisms leading to neuronal cell death in ND.Ferroptosis is a recently recognized form of regulation of cell death,which is different from typical cell death forms?apoptosis,autophagy,necrosis,pyroptosis?,characterized by accumulation of iron-dependent lipid peroxidation.The abnormal increase of reactive oxygen species?ROS?during the occurrence and development of ferroptosis results in the imbalance of intracellular redox.Therefore,we intend to establish an in vitro model with HT-22 cell damage by glutamate,and further study the protective effect of gastrodin and the related mechanism of ferroptosis,so as to provide new therapeutic strategies and methods for the treatment of ND.Purposes:To study the protective effect of gastrodin on ferroptosis induced by glutamate in HT-22 cells.Methods:MTT assay was used to detect HT-22 cells viability,and the optimal treatment time and concentration of gastrodin were determined.The leakage rate of lactate dehydrogenase?LDH?in HT-22 cells was detected by Microenzyme labeling kit.The morphological changes of HT-22 cells were observed by transmission electron microscopy?TCM?;Different inhibitors of Z-VAD-fmk?apoptotic inhibitor?,Necrostatin-1?necrosis inhibitor,Nec-1?,3-Methyladenine?autophagy inhibitor,3-MA?,Ferrostatin-1?ferroptosis inhibitor,Fer-1?and DFO?iron chelator?were detected by MTT assay to observe the effect of gastrodin on glutamate after pretreatment.The activity of SOD in HT-22 cells was detected by WST-1 method;The changes of malondialdehyde?MDA?level in HT-22 cells were detected by thiobarbituric acid?TBA?;Glutathione peroxidase?GPX?activity in HT-22 cells was detected by ultraviolet colorimetry;Flow cytometry was used to detect the changes of ROS and??m;ICP-MS was used to detect the changes of Fe²⁺concentration in HT-22 cell;Changes of related indicators after treatment with Nrf2 inhibitor ML385;The expression of Nrf2,HO-1,GPX4,Ferroportin-1,ACSL4,Ptgs2,Lamin B1 and?-actin protein and the expression of Nrf2 protein after silencing were detected by Western blot.The levels of Nrf2,HO-1 and GPX4 m RNA were detected by RT-qPCR.Results:1.Gastrodin inhibits glutamate-induced HT-22 cells injury?1?MTT assay showed that 5 mM glutamate inhibited HT-22 cells after 24 h of treatment,while 1-25?M gastrodin cells reduced the inhibition of glutamate on HT-22cells;?2?The results of Microenzyme labeling assay showed that LDH leakage rate of HT-22cells treated with 5 m M glutamate was significantly higher than that of normal control group?P<0.01?,while LDH leakage rate of HT-22 cells pretreated with gastrodin was significantly lower than that of glutamate group?P<0.01?,and showed concentration dependence;?3?The results of TCM showed that the mitochondrial ridge was thicker and smaller in the 5 m M glutamate group than in the normal group,while the mitochondrial morphology tended to normal after gastrodin preconditioned HT-22 cells.?4?Z-VAD-fmk?apoptosis inhibitor?,3-MA?autophagy inhibitor?,Nec-1?necrosis inhibitor?,Fer-1?ferroptosis inhibitor?and DFO?iron chelator?were selected as five inhibitors.MTT assay showed that gastrodin pretreated HT-22 cells acted on 5 m M glutamate in a certain concentration range.The inhibition rate of glutamate on cells was decreased?P<0.01?,but Z-VAD-fmk,3-MA and Nec-1 did not?P>0.05?;?5?The results of C11-BODIPY and H₂DCF-DA fluorescence staining showed that gastrodin could inhibit the increase of lipid ROS and cytoplasmic ROS in HT-22 cells induced by glutamate compared with 5 m M glutamate group?P<0.01?;?6?JC-1 fluorescence staining showed that glutamate induced the decrease of mitochondrial membrane potential?MMP?,while gastrodin inhibited the decrease of MMP in a concentration-dependent manner?P<0.01?;?7?ICP-MS assay showed that gastrodin could inhibit the increase of Fe²⁺concentration in HT-22 cells induced by glutamate compared with 5 m M glutamate group?P<0.01?.2.Gastrodin inhibits glutamate-induced HT-22 cells injury through Nrf2/HO-1signaling pathway?1?The Nrf2 nuclear translocation was observed by immunofluorescence,and it was found that gastrodin could promote Nrf2 nuclea translocation;?2?Western blot showed that gastrodin can up-regulate the expression of Nrf2 protein in the nucleus in a time-and concentration-dependent manner;?3?MTT assay showed that the protective effect of gastrodin on glutamate-induced HT-22 cells was inhibited after Nrf2 inhibitor ML385 treatment;?4?Western blot showed that gastrodin had higher expression of Nrf2,HO-1,GPX4 and Ferroportin-1 in HT-22 cells than in glutamate group?P<0.01?,while gastrodin had lower expression of ACSL4 and Ptgs2 in HT-22 cells than in glutamate group?P<0.01?;?5?The results of RT-qPCR showed that the expression of GPX4 m RNA in gastrodin-treated group was higher than that in glutamate-treated group?P<0.01?,but Ptgs2 m RNA decreased?P<0.01?;?6?Western blot showed that the expression of si Nrf2 protein in HT-22 cells treated with gastrodin was inhibited compared with the normal group?P<0.01?;Conclusion:Gastrodin inhibits glutamate-induced ferroptosis in HT-22 cells through Nrf2/HO-1 singnal pathway.