Effect of Nrf2 on brain injury induced by hydraulic shock via regulation of mitophagy and apoptosis

Access to public data

From the GEO (Gene Expression Omnibus) database https://www.ncbi.nlm.nih.gov/gds/ GSE24265 cerebral hemorrhage related data sets were searched and downloaded. R language limma software package was used for GSE24265 data sets quantile standardized preprocessing, and differences in gene analysis (|logFC|<0.5, p-value<0.05), and to get the related differentially expressed genes, and the volcano map and cluster analysis heat map for the differentially expressed genes were drawn.

Functional enrichment analysis

GO (Gene Ontology) enrichment analysis and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis were performed on the common differentially expressed genes obtained from GSE24265 dataset. DAVID (https://david.ncifcrf.gov) online tools were used to GO GSE24265 mRNA differences between genes and KEGG enrichment analysis. GOPlot and ggplot2 package were used to plot the GO pathway and KEGG pathway enrichment analysis of differentially expressed genes in R language environment.

Gene set enrichment analysis

Gene Set Enrichment Analysis (GSEA) tool (http://www.gsea-msigdb.org/) was used for GSEA enrichment analysis of all genes, and GSEA enrichment analysis pathway map was drawn.

Statistical analysis of target genes

Two independent samples t-test were used to compare the two groups of data. GraphPad Prism software was used for drawing. P < 0.05 was considered statistically significant.

Preparation of animal models

Twenty-four Nrf2 knockout (Nrf2-/-) and wild-type mice (WT) of the 5-week-old male C57BL/6J strain were purchased from the Second Hospital of Hebei Medical University. They were placed in a suitable environment (20±2° C, 55±5% relative humidity, and 12/12 h light/dark cycle). They were fed with standard diet and distilled water. KO mice and WT mice were randomly divided into two groups: control group (C) and brain injury group (TBI), with 6 mice in each group (including WT mice, Nrf2-/- mice, WT + fluid percussion brain injury mice, and Nrf2-/- + fluid percussion brain injury mice). This study was approved by the Laboratory Animal Welfare and Ethics Committee of Hebei Medical University, and all protocols followed the ARRIVE (2.0 version) guideline.

Mice in the TBI group received intraperitoneal injection of 2% pentobarbital (100 mg/kg, purchased from Sigma Company, USA), then the skin of the head was prepared and disinfected with iodine volt. The skin and subcutaneous tissue were cut along the median longitudinal, and the periosteum of the skull was cut, 0.4 cm beside the right sagittal suture and 0.4 cm behind the coronal suture as the center point. The bone window with a diameter of about 0.3 cm was drilled with an electric grinding-drill to keep the dura intact and stop the bleeding with bone wax. Mouse head fixed in the fixed frame and adjusted to the appropriate location and height, bone window connection shock tube, denture acrylic bone window close and shock tube connectors, to set the equipment for the hydraulic impact of brain damage strength to 0.05 MPa, trigger switch, high pressure gas drive reservoir tank clean saline impact mice dura mater, completed a potential damage model. Mice were removed, gentamicin saline was used to flush the operative area, bone wax was used to seal the bone window, and scalp incisions were closed. Mice in group C were not treated.

Specimen collection and production

After 24 h of hydraulic shock, the mice were killed by decapitation, and their brains extracted from the ice plate. About 100 mg of brain tissue was taken from the front edge of the injured area, and the water content of brain tissue was measured by dry/wet weight method. The brain tissue at the posterior edge of the injured area was quickly placed in liquid nitrogen, and then rapidly frozen in liquid nitrogen, it was placed in a refrigerator at -80° C for long-term preservation for subsequent experiments.

Hematoxylin-eosin staining (HE)

After the damaged area was cut into coronal slices of about 3 mm and fixed in 4% paraformaldehyde for 24-36 h, dehydration, transparency, paraffin impregnation and embedding were performed according to the conventional procedures. After paraffin was completely dried and fixed, tissue sections were made with a thickness of 4 μm. Brain tissue was stained according to the instructions of HE staining kit. The brain tissue was dewaxed to water, stained with hematoxylin solution for several minutes, rinsed with tap water, divided with 1% hydrochloric acid alcohol for several seconds, rinsed with tap water, then returned to blue with 1% ammonia solution for 1min, rinsed with running water for several seconds, put into eosin staining solution for several seconds, rinsed with running water. Paraffin sections were put into 75% ethanol for 2 min, 85% ethanol for 2 min, anhydrous ethanol for 5 min, anhydrous ethanol for 5 min, xylene for 5 min, and then the sections were sealed with neutral gum from xylene. And the morphological and structural changes of mouse brain tissue were observed under light microscope.

In situ end labeling (TUNEL) analysis

TUNEL assay was used to detect the apoptotic state of brain tissue. Paraffin sections were dewaxed to water according to the conventional method, 1% Triton X-100 at room temperature and washed 3 times with PBS after permeabilization. The tissue sections were covered with protease K working solution and incubated in an incubator at 37° C for 30 min. 50 μL of TdT enzyme reaction solution dropwise was added, and put into a wet box, and react at 37° C for 60 min, avoiding light, and washed with PBS. 50 μL Streptavidin-TRITC labeling solution dropwise was added into a wet box, reacted for 30 min at 37° C, avoiding light, and washed with PBS. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI) and washed with PBS, and observed under fluorescence microscope. The ratio of apoptosis was the number of apoptotic neurons/total number of neurons.

Western blot

RIPA lysate and phenylmethane sulfonyl fluoride (PMSF) were used to extract mouse brain tissue protein samples. Protease inhibitor PMSF was added according to PMSF:RIPA = 1:100 and homogenized on ice. The supernatant was centrifuged at 12000 r at 4° C for 15 min. The supernatant was divided into 200 μL EP tubes and stored at -20° C. BCA kit was used to detect the protein concentration in the supernatant. The loading volume of the sample was calculated according to the protein concentration and loading system of the sample. Ultrapure water and 5× loading buffer were added, and the metal bath was denatured at 100° C for 10 min after mixing and centrifugation. The protein samples were subjected to SDS-PAGE in 5% concentrate gel and 10% separation gel, and the target protein was transferred to PVDF membrane by membrane transfer. Then 5% skim milk powder was sealed at room temperature for 2 hours and incubated overnight with primary antibody at 4° C. After the primary antibody was incubated overnight, the PBST membrane was washed three times for 10 min each time. The membrane was incubated in horseradish peroxidase labeled secondary antibodies at room temperature for 2 h, and then washed 3 times with PBST. Finally, the exposure was performed with the ultra-sensitive ECL chemiluminescence kit and Fluorchem Q instrument, and the protein quantitative analysis was performed with the AlphaView software system.

Extraction and culture of glial cells

Newborn wild type (WT) and Nrf2 gene knockout (KO) suturing mice were killed within 24 hours and soaked in 75% ethanol for disinfection. The meninges and blood vessels were removed. The brain (hippocampus part was removed) was placed in PBS, and the surface blood filaments were rinsed and removed until the brain tissue was milky white. Tissue was torn into chyme with ophthalmic tweezers, and then repeatedly cut with ophthalmic scissors for 10 min. Finally, the tissue was gently blown with pipette gun for 20-30 times. The torn tissue was transferred into a 15 mL centrifuge tube with the gun and digested in a water bath at 37° C for 15 min. Digestion stop solution was added to stop the enzyme reaction. Centrifuged at 1000 rpm for 10 min, supernatant was discarded, culture medium was added to resuspend, and cells were stained and counted. Cells were inoculated at a density of 1×10⁶ cells/mL into culture flask and placed in an incubator at 37° C with 5% CO₂. The appropriate size of the glass slide disinfection treatment was selected, a small amount of culture medium was added to make it adsorbed on the bottom of the six-hole plate. Cells were inoculated and cultured for 24 h, with a density of about 8000 cells per well, and treated in different groups: control group (group C) and brain injury group (TBI group, mechanical pressure was applied to simulate traumatic brain injury).

Preparation of mechanical damage models in vitro

According to Balentine et al., this kind of experimental model was firstly developed to prepare in vitro mechanical compression injury model [18]. A 25-105 mg tailor's needle is dropped directly from a 10 cm high plane onto the exposed surface of the culture, causing the seemingly mature normal culture to suffer temporary shock trauma. Follow-up experiments were performed at a selected post-traumatic time interval (24 h).

Statistical analysis

All data were expressed as mean ± standard deviation $(\overline{\text{x}}\pm \text{s})$, and SPSS 22.0 statistical software (IBM, USA) was used to conduct Student’s t test for the differences in all indicators between groups. Analysis of variance (ANOVA) was adopted for multivariate analysis and SNK was used for post hoc test of differences in means. P<0.05 indicates that the difference is statistically significant.

Histomorphological observation

Under microscope, it was found that the brain tissue structure of all rats in group C was complete, the nerve cells were arranged in order, and the shape was normal. The nucleoli were pale blue and the nuclear membrane was intact, without bleeding, edema and other damage changes. KO mice 24 h after TBI group visible bruises focal point hemorrhage and edema, contusion focal brain tissue around the visible highly porous structure, cell height edema, neuron clearance increases obviously, degeneration of neurons, the cell volume increased obviously, cytoplasm, dye, cell cavity becomes apparent, around with inflammatory cells infiltration and glial cell proliferation. In WT mice, loose tissue structure, mild cell edema, enlarged neuronal space, degeneration of some neurons, and vacuolation in some parts were also observed in the brain tissues around the contusion at 24 h. However, the degree of degeneration and edema in WT mice was significantly less than that in KO mice (Figure 1A).

Figure 1. Histomorphological observation for the brain tissue and TUNEL assay. (A) Histomorphological observation for the brain tissue. (B) Effect of Nrf2 on apoptosis of nerve cells in brain injury induced by hydraulic shock.

Effect of Nrf2 on apoptosis of nerve cells in brain injury induced by hydraulic shock

TUNEL assay was used to detect the apoptotic state of brain tissue, and the TUNEL positive cells were marked as green. There were only a few positive cells in the C group. Compared with C group, the number of TUNEL positive apoptotic cells in the brain tissue around the contusion lesion in TBI group was significantly increased. The apoptosis index of WT mice in TBI group was lower than that in KO mice (P<0.05), the difference was statistically significant (P<0.05) (Figure 1B).

Screening of DEGs

Data set GSE24265 related to cerebral hemorrhage was downloaded from GEO database. Using R language limma package for data set GSE24265 quantile standardized preprocessing, differences in gene analysis (|logFC|<0.5, p-value<0.05), and related differential genes were obtained, including 202 up-regulated genes and 28 down-regulated genes. Volcano maps (Figure 2A) and cluster analysis heat maps (Figure 2B) were drawn for the differential genes.

Figure 2. (A) GSE24265 differential gene volcano map; (B) GSE24265 differential gene cluster analysis heat map; (C, D) GO enrichment analysis of the up-regulated pathway; (E, F) GO enrichment analysis of down-regulated pathway.

Functional enrichment analysis

GO enrichment analysis was performed on DEGS genes using the online tool DAVID, and the up-regulation pathway map of GO was drawn using R language (Figure 2C, 2D), which indicated that the genes were enriched in inflammatory response, immune response, apoptosis pathway, etc. GO down-regulated pathways (Figure 2E, 2F), including transcription, cell adhesion, etc. Differential genes were used to analyze the KEGG pathway and draw the KEGG pathway map, which indicated that the cytokine-cytokine receptor interaction and other related pathways were enriched (Figure 3A). In addition, GSEA gene enrichment analysis revealed enrichment in mitophagy, CASPASE_PATHWAY and other pathways (Figure 3B, 3C).

Figure 3. (A) KEGG pathway enrichment analysis; (B, C) GSEA gene enrichment analysis; (D) The expression analysis of Nrf2.

Statistical analysis of target genes

The content of Nrf2 in different groups was analyzed by statistical methods, and it could be seen that the expression of Nrf2 was low in the disease group and high in the control group (Figure 3D).

Effects of Nrf2 knockout on mitophagy and apoptosis induced by fluid percussion brain injury in vivo

Whether Nrf2 knockout was able to affect the expression of mitophagy-related proteins and apoptosis-related proteins induced by fluid percussion brain injury in vivo was assessed by western blot. Actin was used as a loading control. Bar graphs, representative blots from four independent experiment groups were shown. Representative Western blot images and summarized data showed that Nrf2 knockout decreased the expression of mitophagy-related proteins LC3II, Parkin, Pink and Beclin, while Nrf2 knockout decreased the expression of antiapoptotic protein Bcl-2 and increased the expression of proapoptotic proteins c-caspase-3, c-caspase-8, c-caspase-9, c-caspase-12 and Bax. The data are represented as the mean ± the standard error (SEM). **, P < 0.01. The error bars represent the SEM. Statistical analysis was performed using an unpaired, two-tailed Student's t-test (Figure 4).

Figure 4. (A, B) Effects of Nrf2 knockout on mitophagy and apoptosis induced by fluid percussion brain injury in vivo.

Effects of Nrf2 knockout on mitophagy and apoptosis induced by mechanical pressure in glial cells

Whether Nrf2 knockout was able to affect the expression of mitophagy-related proteins and apoptosis-related proteins induced by mechanical pressure in glial cells was assessed by western blot. Actin was used as a loading control. Bar graphs, representative blots from four independent experiment groups were shown. Representative Western blot images and summary data showed that Nrf2 knockout decreased the expression of mitochondrial autophagy related proteins Lc3II, Parkin, Pink and Beclin in the mechanically damaged mouse model compared with the control group. Knockout of Nrf2 decreased the expression of anti-apoptotic protein Bcl-2, and increased the expressions of mechanical stress-induced pro-apoptotic proteins c-caspase-3, c-caspase-9, c-caspase-12 and Bax. Rapamycin is an inducer of mitophagy. Results showed that Nrf2 knockout did not affect the expression of mitophagy-related proteins and apoptosis-related proteins induced by mechanical pressure and Rapamycin in vivo. The data are represented as the mean ± the standard error (SEM). **, P < 0.01. The error bars represent the SEM. Statistical analysis was performed using an unpaired, two-tailed Student's t-test (Figure 5).

Figure 5. (A, B) Effects of Nrf2 knockout on mitophagy and apoptosis induced by mechanical pressure in glial cells.