Research on the Effects of Neuroglobin on Ferroptosis in the Nerve Cells

Abstract Objectives  The objective of this article was to explore the effects of neuroglobin (NGB) on ferroptosis in the nerve cells. Methods  The NGB knockdown model of HT22 cells was constructed, and the ferroptosis-related indexes of cell proliferation activity, contents of iron ion, malondialdehyde (MDA), superoxide and reactive oxygen, and the changes of nuclear factor E2-related factor 2 (Nrf2) expression were examined in the normal group, erastin group, NGB siRNA group, and NGB siRNA + Erastin group, respectively. Results  Compared with the normal group, cell proliferation activity and Nrf2 expression were significantly lower in the erastin group, NGB siRNA group, and NGB siRNA + erastin group, and ferroptosis-related indexes such as iron ion content, MDA content, superoxide content, and reactive oxygen species content were significantly reduced, and the difference between NGB siRNA + erastin group, erastin group, and NGB siRNA group was statistically significant ( p  < 0.05). Conclusion  Knockdown of NGB in cells enhances the action of the ferroptosis inducer erastin, and NGB may regulate the cellular ferroptosis process through Nrf2. This research may provide references for the clinical treatment of nervous system disease with Chinese medicine.


Introduction
Neuroglobin (NGB) is a globulin mainly expressed in the cytoplasm of nerve cells.2][3] Cerebral hemorrhage belongs to the category of "stroke" in Chinese medicine.Studies have found that the higher the expression level of NGB protein in the brain tissue of patients with cerebral hemorrhage, the better the patient's condition. 4Stroke in Chinese medicine is divided into meridian stroke and visceral stroke according to the state of mind of the stroke patients, and visceral stroke can be divided into obstruction syndrome and collapse syndrome according to the condition of the healthy qi and pathogenic factor.The expressions of NGB in yang obstruction, yin obstruction, and collapse syndrome of stroke are different, which is helpful for the syndrome differentiation and treatment of stroke. 4Chinese medicine has a history of thousands of years and plays an important role in the prevention and treatment of diseases.Studies have found that many Chinese herbs, Chinese compound medicine, and monomer components of Chinese herbs can improve nerve cell damage by regulating the expression of NGB. 5,6In addition, studies have demonstrated that overexpression of NGB can significantly attenuate H 2 O 2 -induced ROS/reactive nitrogen species accumulation and lipid peroxidation, reduce H 2 O 2 -induced mitochondrial dysfunction and apoptosis, which play a protective role against oxidative stress in nerve cells, 7 and polydatin can activate cAMP-response element-binding protein/NGB signaling pathway and protect neuron cells from hydrogen peroxide damage, 8 suggesting that NGB may participate in and improve the oxidative damage of nerve cells, and provide new insights for the research on the mechanism of Chinese medicine in improving the oxidative damage of nerve cells.
Studies have found that the upregulation of NGB expression can promote the survival of nerve cells under oxygen or glucose deprivation and oxidative stress.When the body is in a hypoxic state, the expression of NGB in cells increases rapidly, which is mainly mediated by hypoxia-inducible factor (HIF). 9,10 In 2012, Dixon et al 11 discovered ferroptosis when studying the mechanism of erastin (ferroptosis inducer) leading to the death of RAS mutant tumor cells.Ferroptosis is the regulatory cell death caused by the accumulation of lipid peroxide and ROS due to abnormal iron metabolism in cells.Studies have found that the main mechanism of ferroptosis is divided into three parts: iron metabolism, amino acid metabolism, and lipid peroxidation.9][20][21][22][23][24][25][26][27] In view of the relationship between ferroptosis and HIF, HIF and NGB, and the neuroprotective mechanism of NGB, it is speculated that NGB may participate in cell ferroptosis and improve oxidative damage.Studies have found that the overexpression of NGB can significantly enhance the resistance of human neuroblastoma cells SH-SY5Y to ferroptosis inducers and can effectively protect cells from ferroptosis injury. 28Nuclear factor E2related factor 2 (Nrf2) is a kind of basic leucine zipper (bZIP) transcription factor with anti-oxidative stress effect and it can bind with multiple proteins in cells to exert coregulation effects on multiple genes.Nrf2 is involved in the regulation of various proteins related to iron ion metabolism and can also act on the cystine-glutamate antitransport system and GPX4. 29NGB is also involved in the cystine-glutamate antitransport system. 30Based on the above studies, this experiment constructed the NGB knockdown model of mouse hippocampal neuron HT22 and explored the possible role of NGB in neuronal ferroptosis by examining the changes in related indicators and Nrf2 expression after erastin-induced ferroptosis when NGB was knocked down.

Material Cells
Mouse hippocampal neuron cells (HT22) were donated by West China Second Hospital of Sichuan University.

Drugs and Reagents
Following drugs and reagents were used: NGB siRNA (

Methods
Cell Culture and Grouping HT22 cells were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum and 1 Â 10 5 U/L penicillin-streptomycin under the condition of 37°C and 5% CO 2 .When the confluence of the cells in the culture flask reached 80 to 90%, the cells were subcultured and the cells in the logarithmic growth phase were used for experiments.The HT22 cells were divided into normal group, LipoRNAi group, erastin group, NGBsiRNA interference group and NGBsiRNA þ erastin group.Cells in the normal group were cultured as described above; cells in the LipoRNAi group were cultured in the above manner until the confluence of the cells reached 70 to 80% and, then, replaced with the basal medium containing the corresponding volume of LipoRNAi; cells in the erastin group were cultured as above and replaced with erastin-containing medium (the final concentration of erastin was 20 μmol/L) at corresponding time.In the NGBsiRNA interference group, when the confluence of the cells reached 70 to 80%, they were transfected according to the kit instructions.After transfection, the NGBsiRNA interference group continued to culture as above, while the cells in the NGBsiRNA þ erastin group were replaced with erastin-containing medium (the final concentration of erastin was 20 μmol/L) 24 hours after transfection.

Detection of Neuroglobin Expression Level in Cells
HT22 cells were inoculated at 1.5 Â 10 5 cells/well in a 6-well plate with a clean cover glass, fixed with 4% paraformaldehyde when the confluence of the cells reached 70 to 80%, and Triton-X100 was used to rupture the membrane.Five percent goat serum was used as block solution, NGB polyclonal antibody (1:50 dilution ratio) was added and incubated overnight at 4°C, and goat antirabbit FITC fluorescent secondary antibody (1:600 dilution ratio) was added and incubated at room temperature in the dark for 1 hours and then incubated with DAPI solution at room temperature for 5 minutes; the negative control phosphate-buffered saline (PBS) was used to replace the primary antibody, and the expression of NGB in the cells was observed under an inverted phase-contrast fluorescence microscope and photographed.The cells in the normal group and the NGB siRNA interference group were transfected with NGB siRNA for 48 hours to extract the total cell protein.Protein concentration was determined using the BCA method.Equal amounts of protein samples were mixed with 5Â protein loading buffer at a 4:1 ratio and then subjected to boiling water for 10 minutes to denature the proteins.Subsequently, with a 12% separating gel and a 5% stacking gel in place, the protein samples were sequentially loaded into the sample wells.A constant voltage of 80 V was maintained until the indicator reached the junction of the stacking gel and the separating gel.At this point, the voltage was increased to 100 V for the duration of constant voltage electrophoresis.A polyvinylidene difluoride (PVDF) membrane of 0.22 μm was cut of the same size as the gel and activated in methanol for 10 minutes.Gel and PVDF membrane were placed in the order of cathode to anode, that is, sponge pad-filter paper-gel-PVDF membrane-filter paper-sponge pad, and were rotated at a constant current of 200 mA for 45 minutes.The PVDF membrane was blocked for 1 hour at 37°C in a shaker and washed.NGB (dilution ratio 1:1,000) and beta actin (dilution ratio 1:2,000) were added for incubation in a shaker at 4°C overnight, and the membrane was washed.HRP-labeled goat antirabbit secondary antibody (dilution ratio 1:1,000) was added for incubation in a shaker at room temperature for 1 hour and the membrane was washed.ECL luminescence reagent solution A and solution B were mixed in equal proportions and added dropwise on the PVDF membrane and incubated at room temperature for 3 minutes, and the results were observed in a gel imager, the expression of NGB protein was recorded by taking pictures, and the protein was semiquantitatively analyzed using Image J software.

Detection of Cell Proliferation Activity by the CCK-8 Method
HT22 cells were inoculated in 96-well plates at 1 Â 10 4cells/well, and six replicate wells were set up in each group.Grouping was performed.After incubating in the incubator for 4 hours, the complete medium was used, culturing for 2 hours.Finally, CCK-8 working solution was added, and after incubation in the incubator for 0.5 hours, the absorbance value was detected at 450 nm with a microplate reader, and the measurement was repeated three times.

Detection of Iron Ion Content
HT22 cells were inoculated in 12-well plates at 1.2 Â 10 5 cells/well, and six replicate wells were set up in each group.After being grouped by the method mentioned before, culture continued for 24 hours, cell protein was extracted, and BCA protein concentration was detected.The configuration of the blank tube, standard tube, and test tube reagents followed the instructions provided in the kit.The mixture was placed in a water bath at 100°C for 5 minutes and then cooled with tap water.Subsequently, 60 μL of chloroform was added to each tube, thoroughly mixed, and then centrifuged at 10,000 r•min À1 for 10 minutes.Following centrifugation, 200 μL of the upper inorganic phase was transferred to a 96well plate.Finally, the absorbance at 520 nm was measured using a microplate reader preheated to 37°C, and the iron content for each sample group was calculated.

Malondialdehyde Content Detection
HT22 cells were inoculated in 12-well plates at 1.2 Â 10 5 cells/well, and six replicate wells were set up in each group.After being grouped by the method mentioned before, culture continued for 24 hours, cell protein was extracted, and BCA protein concentration was detected.Complete blank tube, standard tube, and measurement tube reagent configuration were used according to the kit instructions.Each tube was heated in a water bath above 95°C for 40 minutes, cooled under running water, and centrifuged at 4,000 r•min À1 for 10 minutes, and 250 μL of the reaction solution of each tube was pipetted into a 96-well plate, the absorbance value was measured at 530 nm using a microplate reader, and MDA content of each group of samples was calculated.

Superoxide Detection
Cells were inoculated in 6-well plates at 1.5 Â 10 5 cells/well, and three replicate wells were set up in each group.After being grouped by the method mentioned before, the cells were cultured for 4 hours and then replaced with superanion probe working solution (5 mmol/L), 2 mL per well, incubated in an incubator for 30 minutes, washed twice with PBS, and observed and photographed under an inverted fluorescent microscope.

Active Oxygen Detection
Cells were inoculated in 6-well plates at 1.5 Â 10 5 cells/well, and three replicate wells were set up in each group.Following the previously mentioned grouping method, the cells were cultured for 4 hours.Afterward, they were subjected to the DCFH-DA working solution (prepared by diluting the DCFH-DA stock solution with basic culture medium at a ratio of 1:1,000), with 2 mL added per well.The cells were then incubated in a controlled environment for 20 minutes, washed twice with PBS, and subsequently observed and photographed under an inverted fluorescent microscope.

Detection of Nuclear Factor E2-Related Factor 2 Protein Expression
Cells were seeded in 6-well plates at 1.5 Â 10 5 cells/well, and three replicate wells were set up in each group.Cell proteins were extracted 24 hours after adding Erastin, and BCA protein concentration was detected to prepare 10% separating gel and 5% stacking gel.An equal amount of protein sample was taken, mixed with 5Â protein loading buffer in a ratio of 4:1boiled in water for 10 minutes to denature the protein, added well sequentially to the sample, and kept at the voltage constant of 80 V.When the indicator reached the junction of the stacking gel and the separating gel, the voltage was  Gel and PVDF membrane were placed in the order of cathode to anode, that is, sponge pad-filter paper-gel-PVDF membrane-filter paper-sponge pad, and were rotated at a constant current of 200 mA for 1 hour and 20 minutes.The PVDF membrane was sealed for 1 hour at 37°C in a shaker and washed.Nrf2 (1:1,000 dilution ratio) and beta actin (1:2,000 dilution ratio) were added for incubation in a shaker at 4°C overnight, and the membrane was washed.HRP-labeled goat antirabbit secondary antibody (1:1,000 dilution ratio) was added at the room temperature for 1 hour and the membrane was washed.ECL luminescent reagent solution A and solution B were mixed in equal proportions and dropped on the PVDF membrane, incubated at room temperature for 3 minutes; then, the results were observed in a gel imager and the expression of Nrf2 protein was recorded by taking pictures, and the protein was semiquantitatively analyzed using Image J software.

Statistical Analysis
Data analysis was performed using GraphPad Prism 9 software, and the data were expressed as mean AE standard deviation (x̄AE s).The t-test was used for pairwise comparison between groups, and one way analysis of variance was used for data comparison among multiple groups.p < 0.05 indicates that the difference is statistically significant.

Detection Results of Neuroglobin Expression Level in HT22 Cells
Cell immunofluorescence results showed that NGB was expressed in HT22 cells (►Fig.1); compared with the normal group, after NGB siRNA interference, the expression of NGB in HT22 cells in the NGB siRNA interference group was significantly reduced (p <0.05; ►Fig. 2).

Detection Results of CCK-8 Cell Proliferation Activity
Compared with the normal group, the cell proliferation activity of erastin group, NGB siRNA group, and NGBsiRNA þ erastin group was significantly decreased (p < 0.05).There was no significant difference in cell proliferation activity between the erastin group and NGB siRNA group (p > 0.05); compared with cells in the erastin group and NGB siRNA group, the proliferation activity of cells in erastin þ NGB siRNA group was significantly decreased (p < 0.05; ►Fig. 3).

Detection Results of Cellular Iron Ion Content
Compared with the normal group, the iron ion contents in the cells of the erastin group, the NGB siRNA group, and the NGBsiRNA þ erastin group were all significantly increased

Malondialdehyde Content Detection Results
Compared with the normal group, the MDA content in cells of Erastin group, NGB siRNA group and NGBsiRNA þ Erastin group were all significantly increased (p < 0.05); Compared with Erastin group and NGB siRNA group, the MDA content in cells of NGBsiRNA þ Erastin group was significantly increased (p < 0.05; ►Fig. 5).

Detection Results of Superoxide Content
Compared with the normal group, the superoxide content in the cells of the erastin group, the NGB siRNA group, and the NGBsiRNA þ erastin group were all significantly increased (p < 0.05).Compared with the erastin group and NGB siRNA group, the superoxide content in cells of NGBsiRNA þ erastin group was significantly increased (p < 0.05; ►Figs.6 and 7).

Detection Results of Cellular Active Oxygen Content
Compared with the normal group, the content of ROS in the erastin group, NGB siRNA group, and NGB siRNA þ erastin group was significantly increased (p < 0.01).Compared with the erastin group and NGB siRNA group, the content of reactive oxygen in cells of NGB siRNA þ erastin group was significantly increased (p < 0.01; ►Figs.8 and 9).

Nuclear Factor E2-Related Factor 2 Protein Expression Detection Results
Compared with the normal group, the expression of Nrf2 protein in the erastin group, NGB siRNA group, NGB siRNA þ erastin group was significantly decreased (p < 0.05).Compared with the erastin group, there was no significant

Discussion
When ferroptosis occurs in cells, the main morphological manifestations are cell membrane rupture, blebbing, reduction of mitochondrial cristae, mitochondrial miniaturization, and chromatin noncondensation. 31In the process of iron ion metabolism, proteins related to iron absorption and transport, such as transferrin (Tf), transferrin receptor (TfR), and divalent metal transporter 1(DMT1), 14 proteins related to iron storage such as ferritin, 32 and proteins related to iron export, such as iron transporter (ferroportin, Fpn), 33 are abnormally expressed and lead to abnormal accumulation of iron, result-ing in cellular ferroptosis.In terms of amino acid metabolism, ferroptosis is mainly related to GPX4. 34GPX4 is the key enzyme of the important antioxidant system in the human body, which can convert lipid peroxide (ROOH) into nontoxic steroidal alcohol (ROH); thereby, it resists the damage of lipid peroxidation products and ROS to the biomembrane 35 and its antioxidant effect depends on GSH, and the intracellular cystine-glutamate antitransport system is mainly responsible for the synthesis of GSH and further synthesizes GSH by ingesting extracellular cystine and transferring the intracellular toxic glutamate. 36Erastin, a commonly used ferroptosis inducer, acts on the cystine-glutamate antitransport system to cause ferroptosis. 37PUFA provide the molecular basis for the fluidity and deformability of the cell membrane lipid bilayer structure.Due to the presence of carbon-carbon double bonds on PUFA, PUFA in the cell and free radicals will generate peroxidation reaction, which will become the target of ferroptosis. 38When ferroptosis occurs in cells, there will be abnormal accumulation of iron to generate a large amount of ROS, which then reacts with PUFAs to destroy the cell membrane structure and lead to cell death.The above process is accompanied by changes in iron ion content, MDA content, superoxide content, and ROS content, which can be used as the detection index of ferroptosis.
This experiment found that after adding the ferroptosis inducer erastin, the cell proliferation activity decreased, and the contents of cellular iron ions, MDA, superoxide, and cellular ROS increased, indicating that the ferroptosis model of HT22 cells was successfully established.After the knockdown of NGB, the cell proliferation activity decreased, and the contents of cellular iron ion, MDA, and superoxide increased.After the knockdown of NGB, the ferroptosis inducer erastin was added, and the cell proliferation activity was further reduced, and the contents of cellular iron ions, MDA, superoxide, and cellular ROS were significantly increased.The above results indicate that the knockdown of NGB can enhance the effect of erastin and promote ferroptosis.NGB has the function of protecting nerve cells when cranial nerve cells suffer from ischemic hypoxic injury, brain contusion, cerebral hemorrhage, or AD diseases.Studies found that in the mouse model of middle cerebral artery occlusion, NGB overexpression effectively reduced the size of cerebral ischemic infarction, 39 and in the rat model of global cerebral ischemia, limb ischemic preconditioning, the expression of NGB increased, which effectively alleviated the damage of nerve cells caused by ischemia and reperfusion.At the same time, it was also found that the expression level of NGB in the hippocampus, cortex, and hypothalamus was positively correlated with the time of ischemia and reperfusion. 40In the research on AD, it was found that NGB was related to the characteristic changes of AD, such as β-amyloid deposition, hyperphosphorylation of tau protein. 41Simultaneously, the damage caused by β-amyloid deposition in PC12 cells with NGB transfection overexpression was alleviated. 42Xu et al 43 found that 50-Hz high-frequency electroacupuncture stimulation had a better therapeutic effect on cerebral hemorrhage rats than 2 and 15-Hz low-frequency electroacupuncture stimulation, and its therapeutic mechanism may be related to the regulation of NGB expression in hematoma brain tissue.Bushen Yisui decoction combined with acupuncture can significantly increase serum NGB levels in patients with mild cognitive impairment after cerebral infarction and can significantly increase blood flow in ischemic sites and improve cognitive function in patients. 44Tongdu Tiaoshen acupuncture can effectively increase the level of serum NGB in patients with postischemic stroke cognitive impairment on the basis of conventional Western medicine treatment, thereby improving the clinical symptoms of patients. 45The expression of NGB is up-regulated in rats with septic encephalopathy.Electroacupuncture at Baihui (DU 20) and Shuigou (DU 26) in rats has a therapeutic effect on septic encephalopathy, and the effect is most obvious after 24 hours.The action mechanism is related to the up-regulation of NGB expression. 46Yan et al 47 found that acupuncture at Dazhui (DU 14), Baihui(GV 20), and Renzhong (DU 26) could up-regulate the expression level of NGB in the hippocampus of rats after cerebral ischemia-reperfusion injury, thereby achieving brain protection.In an ischemic stroke model, it was found that NGB could effectively remove ROS biomarkers and played a neuroprotective role by combining NO and H 2 O 2 . 48,49Other studies have shown that NGB might exert the ability to remove active substances through the combination of histidine and ROS or through the interaction among the proteins in the respiratory chain. 50,51Based on the above and the results of this experiment, it is speculated that NGB protects neurons not only by acting against cell necrosis and apoptosis 2 but also by inhibiting ferroptosis.
Nrf2 is a key transcription factor that maintains redox balance and normal signal transduction in the human body. 52The six highly conservative functional structure   regions (Neh1-Neh6) can maintain the stability of their own structures and functions and also regulate the expression of related downstream molecules at the transcription level. 48tudies have found that Jinzhan Yinpan (Bidens Biternata) could effectively improve carbon tetrachloride-induced acute liver injury, and the action mechanism was related to the activation of Nrf2/Keap1 signaling pathway to inhibit oxidative stress injury. 53Liuwei Dihuang pill has a significant protective effect on oxidative stress in AD model cells, and its mechanism may be achieved by regulating the expression of antioxidant factors in the Nrf2/HO-1 pathway. 54This experiment found that after NGB expression was knocked down, the expression of Nrf2 in cells decreased, and on the basis of NGB knockdown, erastin could further reduce the expression of Nrf2, indicating that the change of NGB expression can cause the change of Nrf2 expression, and NGB knockdown can enhance the effect of ferroptosis inducer erastin.It is speculated that NGB and Nrf2 might be related to some pathways in the mechanism of ferroptosis.Studies have found that NGB could protect nerves by stabilizing the combination of HIF and DNA, and Nrf2 and DNA.At the same time, NGB could also prevent the abnormal release of iron through HIF and Nrf2 and reduce cell death. 10The steroid hormone 17 β-estradiol (E2) could play an antioxidative stress role through NGB, and in NGB knockout cells, E2's ability to regulate Nrf2 was weakened. 55When studying its neuroprotective effect by constructing manganese porphyrin recombinant metalloprotein (Mn-TAT-PTD-NGB), it was also found that it could effectively clear ROS and restore mitochondrial function.The expressions of Nrf2, superoxide dismutase (SOD), and catalase(CAT) all increased, suggesting that NGB might participate in the body's antioxidative stress process by regulating Nrf2. 56,57

Conclusion
Knockdown of NGB can enhance the effect of iron death inducer erastin and thus promote the occurrence of ferroptosis, and NGB may participate in the process of cell ferroptosis by regulating Nrf2.This study provides a reference for  exploring the mechanism of Chinese medicine to improve the oxidative damage of nerve cells and provides new strategies and inspiration for the clinical application of Chinese medicine in the treatment of neurological diseases.However, due to the complexity of cell injury mechanisms and metabolic pathways, the specific mechanism of NGB involved in the protection of nerve cells against iron death still needs to be further studied.
Effects of Neuroglobin on Ferroptosis in the Nerve Cells Gao et al. e135 increased to 100 V for constant voltage electrophoresis.The PVDF membrane with a pore size of 0.45 μm was cut to match the dimensions of the gel and then activated in methanol for 10 minutes.
Effects of Neuroglobin on Ferroptosis in the Nerve Cells Gao et al.
Effects of Neuroglobin on Ferroptosis in the Nerve Cells Gao et al. e139