Apoptosis Inducible Factor P53 Decrease Glutamate-Associated Damage via Inhibiting Ferroptosis

BACKGROUND: Ferroptosis, a pattern of programmed cell death decided by iron-associated lipid peroxidation, however, its role of p53-mediated xCT pathway in HT22 cell death remains obscure. Herein, this study is to investigate the potential mechanism of the effect of p53-mediated xCT pathway in HT22 cell lines in an iron-relevant mode. METHODS: The viability of HT22 cells were detected by Cell Counting Kit-8(CCK-8) and PI/Hoechst uorescence double staining. The protein expression levels of p53 and xCT were determined by western boltting. DHE uorescence staining technique supervised the intracellular reactive oxygen species (ROS), and intracellular lipid oxidant situation was conrmed by BODIPY 581/591 C11 lipid peroxidation sensor. Intracellular ferrous ions were monitored with FeRhoNox™-1 uoresceent probe. RESULTS: The protein expression levels of p53 was obviously enhanced by tenovin-1 exposure. Accompanied with the upregulation of p53 protein, cell death was decreased signicantly because of glutamate and erastin exposure with 8h, and p53-mediated xCT pathway was activated. Intracelluar ROS levels, lipid oxidant situations and ferrous ions were remarkably restricted from Glutamate-p53 groups in comparison with glutamate groups. CONCLUSIONS: Overall, P53-mediated xCT pathway could decrease the glutamate-associated neurotoxicity, which may be relevant to the inhibition of ferroptosis. Western blot was used to detect the difference of p53 and xCT protein expression. 1 ×10 5 cells / well were vaccinated into 6-well plate for 12 h, then treated with different concentrations of Tenovin-1 (0.1, 1, 10µmol/L) for 6 h, and then the samples were prepared, and the samples were treated with different concentrations of p53 (0.1, 1, 10 µmol/L) for 6 h. The cells were washed twice by PBS and added with lysate containing protease inhibitors and phosphatase inhibitors. The cells were lysed on ice for 10 min, using a cell brush and scraped off the cells and placed in a 1.5 mL EP tube with ultrasound for 3 times for 5 s. BCA protein quantitative kit was used to detect the concentration of protein. The sample was mixed with 3×SDS buffer at 2:1 volume, boiled at 100 ℃ for 5 min, quickly placed on ice to room temperature and centrifuged at 13000 × g for 5 min. After electrophoresis with 35 µg protein sample per well, the protein was transferred to the PVDF membrane at a constant current of 200 mA for 1 h, which was washed 3 times with 5% skimmed milk powder for 1 h. TBST was at room temperature, β-actin antibody was diluted at a dose of 1:1000 each time at room temperature for 1 h and 4 ℃ overnight. After TBS washing 3 times, and 10 min each time. The cells were incubated at room temperature with 1:5 000 dilution for 1 h, developed with ECL kit and β-actin as internal reference; the expression level of xCT protein was detected: 1×10 5 cells per well were inoculated in 6-well plate for 12 h, and the cells were cultured for 12 h. After 8 hours of treatment, the samples were prepared according to the above method, and the expression level of xCT protein was detected by Western blot. XCT and GAPDH antibody were diluted by 1: 1 000.

Page 3/16 intracellular glutathione (glutathione,GSH) deprivation and ROS accumulation (4) . Although Glutamateinduced neuronal death is mainly due to the accumulation of ROS in cells caused by GSH deprivation, it is not the only mechanism. Caspase-dependent apoptosis pathway is involved in the activation of 12lipoxygenase (12-lipoxygenase, 12-lipoxygenase), translocation of apoptosis-inducing factor (apoptosisinducing factor,AIF) and neurotoxicity induced by Glutamate (5) . In recent years, it was found that inhibition of iron death could inhibit glutamate-induced neuronal death in hippocampal slices (3) .
P53 belongs to the p53 family, which mainly regulates cell cycle, induces apoptosis and DNA damage.
p53 plays a key role in neurodegenerative diseases and aging (6) . Some studies have shown that p53 can increase the sensitivity of tumor cells to iron death by regulating the expression of xCT and thus play an anti-cancer role (7) . However, the effect of p53 on iron death in the nervous system is still unclear.
Therefore, we used p53 speci c activator Tenovin-1 in HT22 cell. The model of Glutamate damage was used to induce oxidative stress injury in some neurodegenerative diseases. We hypothesized that ferroptosis was decreased and glutamate-associated neurotoxicity was inhibited by P53-mediated xCT pathway in HT22 cells and its potential mechanism were investigated.

RNA-seq Experiment
Total RNAs was quanti ed by the NanoDrop ND-2000 (Thermo Scienti c) and the RNAs integrity was assessed using Agilent Bioanalyzer 2100 (Agilent Technologies).The sample labeling, microarray hybridization and washing were performed based on the manufacturer's standard protocols. Brie y, total RNAs were transcribed to double strand cDNAs and then synthesized cRNAs. Next, 2nd cycle cDNAs were synthesized from cRNAs. Followed fragmentation and biotin labeling, the 2nd cycle cDNAs were hybridized onto the microarray. After washing and staining, the arrays were scanned by the Affymetrix Scanner 3000 (Affymetrix).

Detection of HT22 cells viability
The viability of HT22 cells was detected by CCK-8 assay. HT22 cells were cultured in 96-well plates at about 1×10 4 / well for 12 h. Glutamate ( nal concentration was 1, 5, 10 mmol/Land Erastin (0.25, 0.5, 11 µmol/L were added. The control group and blank group (corresponding amount of cell culture medium and CCK-8 solution) were set up, each hole was set up 5 compound pores. After 6 hours of incubation in incubator, 10 µL CCK-8 solution was added to each well(100 µL medium per well). After incubated with 2 h in the incubator, the absorbance value of each well was measured at 450 nm wavelength by spectrophotometer, and the relative survival rate of each well was calculated. The relative survival rate = (experimental group -blank group) / (control group -blank group), the experiment repeated 3 times.
The protective effects of p53 activation on glutamate damage and iron death induced by erastin in HT22 cells.
The protective effects of p53 activation on glutamate damage and iron death induced by erastin in HT22 cells were detected by PI/Hoechst uorescence double labeling method. HT22 cells were inoculated with 5×10 4 cells/well in 24-well plate and treated according to above-mentioned method. After treatment for 8 h, the nal concentration of 5 mg/L Hoechst 33342 and PI solution were added to the medium. The cells were incubated with 37℃ and 5% CO 2 for 10 min, and the relative cell survival rate was calculated under uorescence microscope. Relative cell survival rate was expressed as the ratio of Hoechst /(Hoechst + +PI + ) greater than 500 cells. The experiment was repeated three times.
Western blot was used to detect the difference of p53 and xCT protein expression.
Western blot was used to detect the difference of p53 and xCT protein expression. 1 ×10 5 cells / well were vaccinated into 6-well plate for 12 h, then treated with different concentrations of Tenovin-1 (0.1, 1, 10µmol/L) for 6 h, and then the samples were prepared, and the samples were treated with different concentrations of p53 (0.1, 1, 10 µmol/L) for 6 h. The cells were washed twice by PBS and added with lysate containing protease inhibitors and phosphatase inhibitors. The cells were lysed on ice for 10 min, using a cell brush and scraped off the cells and placed in a 1.5 mL EP tube with ultrasound for 3 times for 5 s. BCA protein quantitative kit was used to detect the concentration of protein. The sample was mixed with 3×SDS buffer at 2:1 volume, boiled at 100℃for 5 min, quickly placed on ice to room temperature and centrifuged at 13000 × g for 5 min. After electrophoresis with 35 µg protein sample per well, the protein was transferred to the PVDF membrane at a constant current of 200 mA for 1 h, which was washed 3 times with 5% skimmed milk powder for 1 h. TBST was at room temperature, β-actin antibody was diluted at a dose of 1:1000 each time at room temperature for 1 h and 4℃overnight. After TBS washing 3 times, and 10 min each time. The cells were incubated at room temperature with 1:5 000 dilution for 1 h, developed with ECL kit and β-actin as internal reference; the expression level of xCT protein was detected: 1×10 5 cells per well were inoculated in 6-well plate for 12 h, and the cells were cultured for 12 h. After 8 hours of treatment, the samples were prepared according to the above method, and the expression level of xCT protein was detected by Western blot. XCT and GAPDH antibody were diluted by 1: 1 000.
The lipid oxidation product 4-HNE was detected by immuno uorescence assay.
The HT22 cells were seeded with 5×10 4 cells per well in 24-well plate. Control, P53, Glutamate and Glutamate-p53 group was selected for 8 h according to above-mentioned method. The cells were washed twice with PBS, xed at room temperature with 4% paraformaldehyde for 15 min, washed 3 times with PBS, sealed with 15% donkey serum at room temperature for 1 h, washed with PBS for 3 times, and added with an antibody 4-HNE. The dilution ratio is 1:200, 4℃ overnight after incubation at room temperature for 1 h, washed for 3 times, added with second antibody (dilution ratio of 1:400), incubated at room temperature for 1 h, washed for 3 times, then sealed with 10

Statistical analysis
The statistical analysis was carried out with SPSS 20.0.0 software. The measurement data were expressed as the mean ± SD from at least three biological replicates, and the comparisons between groups was performed by one-way ANOVA (Analysis of Variance), pairwise comparison using Tukey test. A value of p < 0.05 was considered statistically signi cant. Meanwhile, affymetrix GeneChip Command Console (version 4.0, Affymetrix) software was used to extract raw data. Next, Expression Console (version1.3.1, Affymetrix) software offered RMA normalization for both gene and exon level analysis. Then the gene expression analysis and alternative splice analysis proceeded separately. Gene expression analysis: Genesrping software (version 13.1; Agilent Technologies) was employed to nish the basic analysis. Differentially expressed genes were then identi ed through fold change as well as P value calculated with t-test. The threshold set for up-and down-regulated genes was a fold change > = 2.0 and a P value < = 0.05. Afterwards, GO analysis and KEGG analysis were applied to determine the roles of these differentially expressed mRNAs played in these GO terms or pathways. Finally, Hierarchical Clustering was performed to display the distinguishable genes' expression pattern among samples. Alternative splice analysis: Alternative splice analysis was conducted by Transcriptome Analysis Console (version1.0, Affymetrix). Differential exon or junction identi ed through splicing index as well as P value calculated with One-Way Between-Subject ANOVA (Unpaired). The threshold was splicing index > = 2.0 or < = -2.0 Results RNA-seq indicated that p53 participated in glutamateassociated damage in HT22 cells Principal component analysis (PCA): By means of PCA analysis, the distribution of the samples was investigated, the rationality of the experimental design was veri ed, and the homogeneity of the repeated biological samples was demonstrated by two-dimensional diagram( Figure 1A); Cluster analysis: Unsupervised hierarchical clustering of differentially expressed genes was performed(Figure1B); Pathway analysis: The differential genes were analyzed by pathway in KEGG database(Figur1C); Scattered point graph (matrix graph) was shown in Figure 1D.

Tenoin-1 could evoke an increase in the level of p53 protein in HT22 cells
Tenovin-1 induced the increase of p53 protein level in HT22 cells in a concentration-associated manner (included 0.1, 1 and 10 µ mol/L). P53 level in HT22 cells was enhanced at 6 h accompanied with Tenovin-1 exposure, and the p53 expression was increased at 6 h after Tenovin-1 treatment. Therefore, Tenovin-1 was pre-treated for 6 hours. The p53 protein level in HT22 cells treated with 1 µmol/L Tenovin-1 for 6 h was signi cantly higher than that in the control group(P < 0.05). Therefore, 1 µmol/L Tenovin-1 was chose as the optimal concentration to induce p53 activation in subsequent experiments (Fig 2).
P53 inhibited the glutamate-and erastin-associated neurotoxicity of HT22 cells P53 inhibited the glutamate and erastin-associated neurotoxicity of HT22 cells (1,5,10 mmol/L) in a concentration-dependent manner. When the concentration of glutamate was 5 mmol/L, the cell viability of HT22 cells was about 40%. Therefore, 5 mmol/L was chose as the appropriate damage concentration for subsequent experiments. After pretreatment with 1 µmol/L Tenovin-1 for 6 h, the cell death in HT22 cells could be obviously inhibited by glutamate stimulation (P < 0.01) (Fig 3A, 3C). In order to determine whether p53 can decrease iron death, the classical iron death inducer Erastin was used to induce iron death in HT22 cells, and the effect of p53 on the model was detected. Erastin (0.25, 0.5, and 1μmol/L) signi cantly inhibited the cell viability (Fig 3B, 3D). When erastin concentration was 0.5 µ mol/L, the survival rate of HT22 cells was about 35%. Therefore, 0.5 µmol/L was chosen as the suitable concentration for the subsequent toxic test. After exposure to 1 µmol/L Tenovin-1 for 6 h, the cell viability in HT22 cells were signi cantly rescued from erastin-induced cell death by p53 activation (P < 0.01).
P53 inhibited the increase of lipid oxidation in HT22 cells activated by glutamate.
After 8 h of glutamate exposure, the lipid oxidation level of HT22 cells was increased about 4 times compared with the control group, howerer, in Glu-p53 group, the level of lipid oxidation decreased signi cantly (P <0.01). After lipid oxidation, 4-HNE content (the product of cell lipid metabolism) of the cells was increased signi cantly (Fig 4A). Then glutamate was treated for 8 h, we found that the 4-HNE content was signi cantly higher than that of the control group (P <0.01). However,the level of 4-HNE in glutamate-p53 group was signi cantly lower than that in control group (P<0.01) (Fig 4B).

P53 inhibited the increase of intracellular iron ion in HT22 cells activated by glutamate
The concentration of divalent iron ion was increased by about 4 times in HT22 cells in glutamate group compared with the control group (P < 0.01), the intracellular iron concentration of HT22 in Glutamate-p53 group was signi cantly lower than that in control group (P <0.01) (Fig 5), which demonstrated that activation of p53 remarkably inhibited the increase of iron ion concentration induced by glutamate.

P53 reversed the inhibition of xCT expression induced by Glutamate and Erastin in HT22 cells
The expression level of xCT protein was decreased in glutamate group(P < 0.05) compared with the control group (Fig 6A). The expression of xCT protein in glutamate-p53 group was signi cantly higher than that in glutamate group (P < 0.05). In the classical iron death inducer Erastin damage model ( Fig  6B), the expression of xCT protein was signi cantly inhibited in erastin group in comparison to the control group(P < 0.01), and which was also signi cantly increased in erastin-p53 group than that in erastin group (P < 0.01).

Dicussion
Iron death is a regulatory necrosis characterized by lipid oxidation and iron dependent pathway, which is different from traditional caspase-dependent apoptosis and cell necrosis. In recent years, studies have shown that iron death may be inextricably linked to Parkinson's disease, Alzheimer's disease, and Huntington's disease (8)(9)(10) .
Some studies have shown that iron death is closely related to p53 activation in lung cancer and osteosarcoma, and this process mainly depends on the direct transcription inhibition of xCT (The main component of System Xc) (11) . Our results showed that p53 not only did not increase the sensitivity of glutamate-associated damage in HT22 cells, but also rescued cell viability from HT22 cell death. And we carried out further research to make sure the role of iron death.
Erastin, a classical iron death inducer, can induce iron death. We used tenvion-1 to pretreat with HT22 cells for 6 h to induce p53 activation, and then erastin was administrated to induce iron death. Our results showed that the survival rate was signi cantly higher in erastin-p53 group than that of erastin group, which indicated that p53 pathway could restain from the iron death of HT22 cells. In order to verify whether p53 inhibits iron death in the glutamate-induced damage model and thus protect HT22 cells from glutamate-evoked neurotoxicity, we further explore the cell survival rate in T22 cells exosure to glutamate in the situation of both p53 activation and no p53 activation. It was found that the levels of ROS, lipid oxidation and divalent iron ion in glutamate-p53 group were signi cantly lower than those in glutamate group, which suggested that p53 could inhibit the iron death of HT22 cells and make HT22 cells exempt from the neurotoxicity of glutamate.
Since studies have shown that p53 increases the sensitivity of tumor cells to iron death in lung cancer and osteosarcoma cells may be due to the regulation of xCT protein expression (6) . In order to explore the possible mechanism of p53 resistance to iron death, we examined the expression of xCT protein in HT22 cells and found that the expression of xCT protein was inhibited in glutamate group and erastin group, which was also con rmed in the previous study (12) . In glutamate-p53 and erastin-p53 groups, the inhibition of xCT protein expression was reversed in a certain extent. It is suggested that xCT plays a key role in the inhibition of glutamate-and erastin-associated damage by p53 acitivation.
In our study, the role of p53 in neurodegenerative diseases and iron death was investigated mainly on HT22 cells. HT22 cell, a mouse hippocampal neuron line, which derived from HT4 cell lines, and adherent to the growth under normal conditions. Because of the lack of ion channel glutamate receptor, it is suitable to study the oxidative stress damage model of Glutamate, which is widely used in many degenerative diseases researches. However, there were some limitations, HT22 cell line is different from primary nerve cells and animal neurons, so the role of p53 in primary nerve cells and animals needs to be further excavated; Meanwhile, xCT is not silenced and the protective effect of p53-mediated xCT pathway is needed to further explored.
In conclusion, this study suggests that p53-mediated xCT pathway can protect neural celsl from the neurotoxicity of glutamate by inhibiting iron death, which provides a novel strategy to excavate the role and potential mechanism of p53 in many neurodegenerative diseases. RNA-seq information in HT22 cells The samples of the same group were concentrated in the twodimensional spatial distribution, indicating that the selection of these genes was representative and the biological duplication was good( Figure 1A); The distance between the two pairs of samples is calculated to form a distance matrix. The two nearest classes of distance are combined into a new class, and the distance between the new class and the current class is calculated, then merged and calculated until there is only one class. The expression of selected differentially expressed genes was used to calculate the direct correlation of the samples. In general, the same samples can be clustered in the same cluster, and the genes clustered in the same cluster may have similar biological functions. Display with Heatmap (Figure1B). The signi cance of gene enrichment in each pathway entry was calculated by statistical test. The calculated results return a signi cantly enriched P value, and a small p value indicates that the differential gene is enriched in the pathway. We can nd out that p53 pathway may be related to the differentially expressed genes in different samples (Figur1C). The scatter plot of chip data is often used to evaluate the trend of two sets of data distribution centralization ( Figure 1D).

Figure 2
Tenovin-1 induced protein levels of p53. P53 were detected and quanti ed relative to GAPDH by western blot analysis. And relative amount of expression of p53 protein was statistically analyzed in HT22 cells(included 0.1, 1 and 10 µ mol/L). The p53 protein level in HT22 cells treated with 1 µmol/L Tenovin-1 for 6 h was signi cantly higher than that in the control group( Figure 2). Error bars: standard error of mean. Data were obtained from at least three separate cultures, given as mean ± SEM, and analyzed by two-way ANOVA followed by Bonferroni multiple comparison tests . *P<0.05 vs control.  p53 decreased intracellular lipid oxidation evoked by glutamate. Fluorescence intensity of lipid oxidation in HT22 cells was decreased by p53 compared with glutamate group (##P<0.01 vs Glu.)( Figure 4A).
Meanwhile, glutamate could increase the intracellular lipid oxidation. The average uorescence intensity of each group was calculated; The lipid oxidation product 4-HNE was detected by immuno uorescence assay in HT22 cells ( Figure 4B). p53 decreased expression of 4-HNE in HT22 cells induced by glutamate ( Figure 4B) (scale bar=100 μm). Error bars: standard error of mean. Data were obtained from at least three separate cultures, given as mean ± SEM, and analyzed by two-way ANOVA followed by Bonferroni multiple comparison tests . **P<0.01 vs control; ##P<0.01 vs glutamate, respectively. Figure 5 p53 reduced intracelluar ferrous level in HT22 cells activated by glutamate. FeRhoNox-1 uorescent probe was used to detect the changes of intracellular iron ions in HT22 cells in control group, p53-H group, glutamate group and glutamate-p53 group. The average uorescence intensity of each group was calculated, which indicated that p53 reduced intracelluar ferrous level in HT22 cells activated by glutamate( Figure 5). Error bars: standard error of mean. Data were obtained from at least three separate cultures, given as mean ± SEM, and analyzed by two-way ANOVA followed by Bonferroni multiple comparison tests . **P<0.01 vs control; ##P<0.01 vs Glu, respectively. Figure 6 p53 reversed from suppression of the expression of xCT protein in HT22 cells acitivated by glutamate and erastin. p53 were detected and quanti ed relative to β-actin by western blot analysis. And relative amount of expression of xCT protein was statistically analyzed in HT22 cells in control group, p53-H group, glutamate group and glutamate-p53 group, which indicated that p53 increased expression of xCT in HT22 cells reduced by glutamate. Error bars: standard error of mean( Fig 6A). p53 were detected and quanti ed relative to β-actin by western blot analysis. And relative amount of expression of xCT protein was statistically analyzed in HT22 cells in control group, p53-H group, era group and era-p53 group, which indicated that p53 increased expression of xCT in HT22 cells reduced by erastin (Fig 6B). Error bars: standard error of mean. Data were obtained from at least three separate cultures, given as mean ± SEM, and analyzed by two-way ANOVA followed by Bonferroni multiple comparison tests . #P<0.05 vs Glu; **P<0.01vs CON, respectively.