Dl-3-n-butylphthalide exerts neuroprotective effects by modulating hypoxia-inducible factor 1-alpha ubiquitination to attenuate oxidative stress-induced apoptosis

Abstract

Dl-3-n-butylphthalide is used to treat mild and moderate acute ischemic stroke. However, the precise underlying mechanism requires further investigation. In this study, we investigated the molecular mechanism of Dl-3-n-butylphthalide action by various means. We used hydrogen peroxide to induce injury to PC12 cells and RAW264.7 cells to mimic neuronal oxidative stress injury in stroke in vitro and examined the effects of Dl-3-n-butylphthalide. We found that Dl-3-n-butylphthalide pretreatment markedly inhibited the reduction in viability and reactive oxygen species production in PC12 cells caused by hydrogen peroxide and inhibited cell apoptosis. Furthermore, Dl-3-n-butylphthalide pretreatment inhibited the expression of the pro-apoptotic genes Bax and Bnip3. Dl-3-n-butylphthalide also promoted ubiquitination and degradation of hypoxia inducible factor 1α, the key transcription factor that regulates Bax and Bnip3 genes. These findings suggest that Dl-3-n-butylphthalide exhibits a neuroprotective effect on stroke by promoting hypoxia inducible factor-1α ubiquitination and degradation and inhibiting cell apoptosis.

Introduction

Stroke is the second leading cause of death and disability worldwide, with ischemic cerebrovascular disease accounting for 80% of all strokes (Koh and Park, 2017). Thrombolytic therapy and arterial intervention are the most effective treatment methods for stroke (Brott et al., 1994). These methods are aimed at restoring the blood supply to the ischemic area caused by the stroke as soon as possible (Prabhakaran et al., 2015). However, calcium overload (Zhou et al., 2021) and oxidative stress (Deng et al., 2021; Jiang et al., 2022; Ma et al., 2022; Zhu et al., 2022) after stroke can lead to a cascade of pathologies, subsequently resulting in neuronal necrosis and blood-brain barrier (BBB) damage (Jiang et al., 2018). Reducing oxidative stress injury is a key part of the treatment of ischemic stroke and a priority in research.

Dl-3-n-butylphthalide (NBP) is a small molecule drug used to treat mild to moderate acute ischemic stroke (Huang et al., 2018; Wang et al., 2018; Chen et al., 2019; Fan et al., 2021). NBP has been reported to inhibit apoptosis after ischemic injury (Liu et al., 2021). Another study found that NBP treatment promotes neurological functional recovery in a cerebral ischemia mouse model (Wang et al., 2019). The protective effects of NBP on vascular endothelial cells, BBB and neurons suggest the potential of NBP in rescuing brain function after ischemic injury (Li et al., 2019a). However, its precise role and molecular mechanism have not been completely elucidated.

In this study, we investigated the neuroprotective effects of NBP in response to oxidative stress injury by simulating oxidative stress injury in neuronal cells with H₂O₂ treatment. We further explored the potential molecular mechanism of NBP.

Methods

Cell culture

PC12 (Cat# TCR9) and RAW 264.7 (Cat# TCM13) cells, which are common in vitro models for neurological diseases and macrophage research, were purchased from the Chinese Type Culture Collection, Chinese Academy of Sciences (Shanghai, Chinese). Cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Biological Industries, Jerusalem, Israel) in a humidified atmosphere at 37°C containing 5% CO₂. To establish an oxidative stress injury model, PC12 cells (1 × 10⁴ cells/well) were inoculated in 96 well plates and treated with a final concentration of 200 µM H₂O₂ (Sigma, St. Louis, MO, USA) for 4 hours. The cells were pretreated with 20 and 40 µM NBP (Shiyao Pharmaceutical Co., Ltd., Shijiazhuang, Chinese) for 24 hours. Untreated cells served as controls.

Cell viability analysis

Cells were incubated with Cell Counting Kit-8 solution (CCK8, 10 µL; MCE, Shanghai, China) for 60 minutes. Absorbance was measured at 450 nm using a spectrophotometric microplate reader (Gene Company Ltd., Shenyang, China).

5-Ethynyl-2’-deoxyuridine incorporation assay

PC12 cells (1 × 10⁴ cells/well) were inoculated in appropriate numbers into 96-well plates and treated as indicated. Viability was evaluated using the BeyoClick™ EdU Cell Proliferation Kit (APExBIO, Houston, TX, USA). EdU pre-warmed at 37°C was added to 96-well plates and cells were then examined following the kit instructions. Cells were observed and photographed using a fluorescent inverted microscope (Leica, Wetzlar, Germany).

Caspase-3 activity assay

Caspase-3 activity was measured using GreenNuc Caspase-3 substrate (Beyotime, Shanghai, China) following the manufacturer’s protocol. In brief, PC12 cells were plated into 96 wells (5 × 10⁴ cells/mL) and treated as indicated. After washing cells twice with phosphate buffered saline (PBS), GreenNuc Caspase-3 substrate was added to each well and cells were incubated for 30 minutes. Hoechst 33342 solution (Beyotime) was then added to each well and plates were incubated for 10 minutes at 18–26°C and protected from light. Cells were analyzed under a fluorescence microscope (Leica).

Mitochondrial membrane potential assay

PC12 cells were treated with 5 μM tetramethylrhodamine ethyl ester (TMRE, MCE) at 37°C for 15 minutes; the supernatant was removed and cells were washed twice with pre-warmed cell culture medium to remove excess dye. After Hoechst 33342 staining, photographs were taken under a fluorescence microscope.

Intracellular reactive oxygen species assay

Intracellular reactive oxygen species (ROS) production was detected using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA; MCE). Briefly, after NBP treatment, PC12 cells were washed with prewarmed PBS and stained with 10 µM DCFH-DA for 20 minutes at 37°C. Cells were washed three times with pre-warmed cell culture medium to remove excess dye and subjected to H₂O₂ treatment for 4 hours. Hoechst 33342 was used to stain cells for 10 minutes, and images were acquired with a fluorescence microscope.

Transcriptome sequencing and differentially expressed gene analysis

Total RNA was extracted from PC12 cells, and whole transcriptome sequencing was sequenced on an Illumina Novaseq platform and bioinformatics data were analyzed by Origin-Gene Company (Shanghai, China). The differentially expressed genes (DEGs) results were analyzed in the RNA sequencing results after transcriptome sequencing. Regulatory interactions were examine between transcription factors and target genes using the TRRUST database (https://www.grnpedia.org/trrust/) (Han et al., 2018). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of the DEGs were conducted by clusterProfiler package (version 3.14.3; https://bioconductor.org/packages/release/bioc/html/clusterProfiler.html).

Real-time polymerase chain reaction

Trizol reagent (Thermo Fisher, Waltham, MA, USA) was used to isolate RNA from PC12 and RAW264.7 cells. cDNA was synthesized from 3 µg of RNA template with the RevertAid Master Mix Reagent (Thermo Fisher) following the manufacturer’s instructions. Real-time polymerase chain reaction (qPCR) was performed using Platinum SYBR Green qPCR SuperMix (Thermo Fisher). Gene expression was calculated using the ΔΔCt method (Livak and Schmittgen, 2001); glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as the internal control. Primer sequences were as follows: Bax: forward: 5′-AGA CAG GGG CCT TTT TGC TAC-3′, reverse: 5′-AAT TCG CCG GAG ACA CTC G-3′; Bnip3: forward: 5′-CTG GGT AGA ACT GCA CTT CAG-3′, reverse: 5′-GGA GCT ACT TCG TCC AGA TTC AT-3′; Mmp-2: forward: 5′-CAA GTT CCC CGG CGA TGT C-3′, reverse: 5′-TTC TGG TCA AGG TCA CCT GTC-3′; Mmp-9: forward: 5′-CTG GAC AGC CAG ACA CTA AAG-3′, reverse: 5′-CTC GCG GCA AGT CTT CAG AG-3′; and GAPDH: forward: 5′-AGG TCG GTG TGA ACG GAT TTG-3′, reverse: 5′-GGG GTC GTT GAT GGC AAC A-3′.

Immunoprecipitation

PC12 cells were incubated with 10 µM MG132 (Beyotime) for 9 hours. Immunoprecipitation was performed using an anti-hypoxia-inducible factor 1-alpha antibody (HIF-1α; rabbit, 1:100, Proteintech, Chicago, IL, USA, Cat# 20960-1-AP, RRID: AB_10732601). Immunoprecipitated proteins were subjected to western blot as described below.

Western blot assay

The PC12 cells were lysed using mixed cell lysates containing protease inhibitors at 4°C for 30 minutes, and 12,000 × g for 15 minutes, and the supernatant was collected. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. After blocking, the membranes were probed with anti-HIF-1α and anti-GAPDH (1:1000, Proteintech, Cat# 10494-1-AP) overnight at 4°C. For immunoblotting of immunoprecipitation complexes, membranes were incubated with the ubiquitin primary antibody (mouse, 1:1000, Proteintech, Cat# 10201-2-AP, RRID: AB_671515) overnight at 4°C. Membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (mouse, 1:1000, Abcam, Cambridge, MA, USA, Cat# ab131366, RRID: AB_2892718) for 1 hour at room temperature. Protein bands were visualized by electrochemiluminescence (Beyotime) using Quantity One (Bio-Rad).

Chromatin immunoprecipitation

PC12 cells were incubated in 1% formaldehyde for 15 minutes and cells were lysed using mixed cell lysates at 4°C for 30 minutes. DNA was digested with micrococcal nuclease (NEB, Ipswich, MA, USA). After the addition of chromatin immunoprecipitation dilution buffer (Abcam, Cambridge, MA, USA), part of the sample (50 µL) was saved as input. Samples (500 µL) were then incubated with HIF-1α antibody overnight at 4°C. Protein A/G magnetic beads (MCE) were added to the antibody/chromatin complex and the mixture was incubated at room temperature for 2 hours. The complexes were resuspended in wash buffer, and the protein/DNA cross-links were cleaved to obtain free DNA. The purified DNA was resuspended in tris-ethylenediaminetetraacetic acid buffer (Sango Biotech, Shanghai, China) for polymerase chain reaction.

Matrix degradation assay

Glass coverslips were coated with 0.2 mg/mL of fluoresceine isothiocyanate-gelatin (Invitrogen, Carlsbad, CA, USA) and cross-linked with 0.5% glutaraldehyde. Coverslips were incubated in 10% fetal calf serum-containing medium overnight at 37°C. RAW 264.7 cells were cultured on the coated coverslips for 8 hours. Cells were stained with phalloidin (Beyotime), and nuclei were stained with 4′,6-diamidino-2-phenylindole (Beyotime). Cells were examined under a confocal microscope (Leica TCS SP8).

Statistical analysis

Statistical analyses were performed using GraphPad Prism 6.01 statistical software (GraphPad Software, San Diego, CA, USA, www.graphpad.com). Comparisons between groups were made using one-way analysis of variance followed by Bonferroni correction. P < 0.05 was considered statistically significant.

Results

NBP improves cell viability and inhibits apoptosis in H₂O₂-treated cells

PC12 cells are commonly used as an in vitro model to study central nervous system neurons (Wu et al., 2020). We first examined the effects of NBP on the viability of PC12 cells treated with H₂O₂, as a model of oxidative stress, using CCK8. While H₂O₂ reduced the viability of cells compared with controls, NBP pretreatment significantly prevented the decrease in viability of H₂O₂-treated cells (P = 0.0344; Figure 1A). EdU assay showed that NBP pretreatment reversed the inhibition of cell proliferation after H₂O₂ treatment (Figure 1B). Caspase-3 plays a key role in regulating apoptosis (Porter and Jänicke, 1999). Caspase-3 activity assays showed that pretreatment with NBP significantly reduced the induction of caspase-3 activity caused by H₂O₂ treatment (Figure 1C).

Figure 1.

NBP improves cell viability and inhibits apoptosis in H₂O₂-treated cells.

(A) PC12 cells were treated with 200 µM H₂O₂ for 4 hours. The cells were pretreated with 20 and 40 µM NBP for 24 hours. CCK8 assay was used to explore the effect of NBP on cell viability. (B) PC12 cells were treated with 200 µM H₂O₂ for 4 hours. The cells were pretreated with 40 µM NBP for 24 hours. EdU was used to examine the effects of NBP on cell proliferation (original magnification, 100×). (C) Caspase-3 activity assay in PC12 cells (original magnification, 100×). Data are presented as mean ± SD. The experiment was repeated three times. *P < 0.05, **P < 0.01, ****P < 0.0001 (one-way analysis of variance followed by Bonferroni correction). CCK8: Cell Counting Kit-8; CTL: control; EdU: 5-ethynyl-2′-deoxyuridine; NBP: Dl-3-n-butylphthalide.

NBP mitigates H₂O₂-induced mitochondrial dysfunction and ROS contents in PC12 cells

To assess whether NBP protected PC12 cells against H₂O₂ treatment by mitigating mitochondrial dysfunction associated with apoptosis, we measured the mitochondrial membrane potential by TRME staining assay. TRME intensity decreased following H₂O₂ exposure; however, NBP cotreatment prevented H₂O₂-induced changes in the mitochondrial membrane potential (Figure 2A). These results demonstrated that NBP mitigated H₂O₂ exposure-induced mitochondrial dysfunction.

Figure 2.

NBP protects mitochondrial function and inhibits ROS production in PC12 cells exposed to H₂O₂.

(A) PC12 cells were treated with 200 µM H₂O₂ for 4 hours. The cells were pretreated with 40 µM NBP for 24 hours. Mitochondrial membrane potential was determined by evaluation of TMRE staining under a fluorescence microscope (100× magnification). (B) Detection of ROS by DCFH-DA staining in PC12 cells (100× magnification). ROS levels were increased after H₂O₂ exposure compared with controls, and NBP pretreatment inhibited the increase in ROS levels. The experiment was repeated three times. CTL: Control; DCFH-DA: 2,7-dichlorodi -hydrofluorescein diacet; NBP: Dl-3-n-butylphthalide; ROS: reactive oxygen species; TRME: N,N,N’,N’-tetra-methylethylenediamine.

We next investigated the effects of NBP on intracellular ROS levels in PC12 cells after H₂O₂ treatment using DCFH-DA. The results showed that ROS levels increased after H₂O₂ exposure compared with controls, and NBP pretreatment inhibited the increase in ROS levels (Figure 2B).

Transcriptome sequencing to obtain DEGs and enrichment analysis

We next performed transcriptome sequencing analysis of H₂O₂-treated cells, NBP pretreated and H₂O₂-treated cells and control cells and identified DEGs. The overall distribution of the DEGs was represented by volcano plots (Figure 3A and B). The results identified 1338 DEGs in the H₂O₂ group compared with the control group (722 up-regulated and 616 down-regulated DEGs) (Figure 3A) and 1233 DEGs were found in the NBP + H₂O₂ group compared with the H₂O₂ group (643 up-regulated and 590 down-regulated DEGs) (Figure 3B). We then obtained 474 overlapping DEGs between the two sets of DEGs (Figure 3C).

Figure 3.

Identification of DEGs from transcriptome sequencing analysis.

(A) PC12 cells were treated with 200 µM H₂O₂ for 4 hours. The cells were pretreated with 40 µM NBP for 24 hours. Volcano plot visualizing DEGs between H₂O₂-treated and control PC12 cells. (B) Volcano plot of the DEGs between NBP-pretreated H₂O₂-treated and H₂O₂-treated cells. (C) Venn diagram showing the overlapping DEGs. CTL: Control; DEG: differentially expressed gene; NBP: Dl-3-n-butylphthalide.

Enrichment analysis was performed to analyze the functions of the overlapping DEGs. KEGG pathway analysis revealed that the DEGs were mainly enriched in phosphatidylinositol 3 kinase (PI3K)/protein kinase B (AKT), apoptosis and cellular senescence signaling pathways (Figure 4A). GO functional analysis revealed that DEGs were mainly enriched in biological processes of mRNA processing (Figure 4B). GO functional analysis revealed that the DEGs were enriched in nuclear speck and chromatin binding in the top 10 enrichment classes (Figure 4C and D).

Figure 4.

GO and KEGG pathway enrichment analyses of the overlapping DEGs.

(A) Bubble plot of KEGG pathway enrichment analysis of the DEGs. (B) Bubble plot of biological process enrichment analysis of the DEGs. (C) Bubble plot of cellular component enrichment analysis of the DEGs. (D) Bubble plot of molecular function enrichment analysis of the DEGs. DEG: Differentially expressed gene; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; NBP: Dl-3-n-butylphthalide.

NBP promotes HIF-1α degradation and may affect target gene transcription to inhibit apoptosis

Mitochondria play an important role in regulating caspase activity to promote apoptosis. Our results showed that NBP affects the mitochondrial membrane potential and ROS production in PC12 cells, which in turn inhibits apoptosis. Enrichment analysis of the DEGs obtained by transcriptome sequencing revealed that the overlapping DEGs were enriched in the endogenous apoptotic pathway (Additional Table 1), suggesting that NBP may regulate apoptosis by affecting the expression of these genes. The results of the enrichment analysis showed that Bcl2l1, Bax and Bnip3 genes were enriched in the apoptotic signaling pathway in PC12 cells. Therefore, we examined the expression levels of these genes in PC12 cells by real-time PCR. The results showed that H₂O₂ upregulated the expression of Bax and Bnip3 genes compared with controls, while NBP reversed this effect (Figure 5A).

Additional Table 1.

List of 31 differentially expressed genes involved in apoptosis

ID	Description	Gene ratio	P-value	FDR	Gene ID	Size
GO:200 1233	Regulation of apoptotic signaling pathway	23/40 6	4.07E-0 5	0.005 724	Ybx3/Selenos/Rffl/Bcl2l1/Pten/Eno1/Rpl26/Vdac2/Bax/Gsn/Becn1/Arhgef2/Eif2ak3/M ff/Slc35f6/Bnip3/Rps7/Tfdp1/Gnai2/Ywhaq/Ywhah/Ctnnb1/Raf1	23
GO:009 7193	Intrinsic apoptotic signalingpathway	15/40 6	0.00171 2	0.047 472	Ybx3/Selenos/Bcl2l1/Eno1/Rpl26/Vdac2/Bax/Becn1/Arhgef2/Eif2ak3/Aifm1/Bnip3/Br sk2/Rps7/Stk25	15

FDR: False discovery rate.

Figure 5.

NBP promotes HIF-1α ubiquitination in H₂O₂-treated cells.

(A) PC12 cells were treated with 200 µM H₂O₂ for 4 hours. The cells were pretreated with 40 µM NBP for 24 hours. The mRNA expression of Bnip3 and Bax were detected by real-time polymerase chain reaction. GAPDH mRNA was used as the internal control. Data normalized by control group are presented as mean ± SD. The experiment was repeated three times. **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Bonferroni correction). (B) Analysis of HIF-1α ubiquitination (Ub) by immunoblot analysis. (C) Chromatin immunoprecipitation-polymerase chain reaction analysis of HIF-1α binding to Bnip3 and Bax promoters using antibodies against HIF-1α in PC12 cells. Bax: BCL2-associated X; Bnip3: BCL2 interacting protein 3; CTL: control; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HIF-1α: hypoxia-inducible factor 1-alpha; IP: immunoprecipitation; NBP: Dl-3-n-butylphthalide.

The TRRUST database can be used to predict the regulatory interactions between transcription factors and target genes (Han et al., 2018). We used TRRUST to predict transcription factors of the DEGs involved in regulating the apoptotic process (Additional Table 2), and the results showed that HIF-1α was the key transcription factor regulating the DEGs.

AdditionalTable 2.

Predicting transcription factors of differentially expressed genes

Key transcription factor	Numbers of genes	P-value	Q-value	List of overlapped genes
HIF1A JUN	3 2	3.59E-05 0.00608	0.00018 0.0122	Bax, Bnip3, Eno1 Bcl2l1, Becn1
TP53	2	0.00732	0.0122	Bax, Bcl2l1
RELA	2	0.0232	0.0235	Bcl2l1, Bax
NFKB1	2	0.0235	0.0235	Bax, Bcl2l1

We next examined the effect of NBP on HIF-1α protein expression after H₂O₂ treatment in PC12 cells by western blot analysis. The results showed that HIF-1α protein increased after H₂O₂ treatment compared with controls and it was down-regulated in the NBP pretreatment group compared with the H₂O₂ treatment group (Figure 5B). However, the HIF-1α gene was not identified as a DEG in transcriptome sequencing analysis. The alteration of HIF-1α protein levels but not mRNA levels in response to NBP may involve modulation of steady state protein levels through ubiquitination-mediated modification. Thus, we next analyzed the ubiquitination level of HIF-1α. While HIF-1α was non-ubiquitinated under H₂O₂ conditions, NBP promoted HIF-1α ubiquitination (Figure 5B).

To confirm that Bnip3 and Bax genes are transcriptionally regulated by HIF-1α, we performed chromatin immunoprecipitation assays. The results showed that HIF-1α may bound to Bnip3 and Bax promoters (Figure 5C).

Together these results suggest that NBP treatment results in the ubiquitination and subsequent degradation of HIF-1α under H₂O₂ conditions, leading to inhibition of the transcription of HIF-1α downstream genes.

NBP inhibits H₂O₂-induced activation of macrophage-derived matrix metalloproteinases

BBB damage is a critical pathological process that contributes to poor prognosis in cerebral ischemia (Li et al., 2019b), inhibition of matrix metalloproteinases protects the BBB (Li et al., 2019b; Candelario-Jalil et al., 2022). Given the reported protective effects of NBP on the BBB (Li et al., 2019a; Wu et al., 2020), we determined the effect of NBP on extracellular matrix (ECM) degradation in H₂O₂-treated RAW 264.7 macrophages, which have been reported as a potential source of MMPs that can degrade the BBB (Jiang et al., 2018). Matrix degradation assays showed that H₂O₂ treatment caused ECM degradation around the periphery of macrophages, and this was attenuated by NBP (Figure 6A). We next sought to investigate the mechanisms by which NBP treatment blocked H₂O₂-induced BBB degradation. Real-time PCR of RAW 264.7 macrophages demonstrated that H₂O₂ increased Mmp-2 and Mmp-9 mRNA expressions compared with controls, while NBP blocked the increase of Mmp-2 and Mmp-9 mRNA expression (Figure 6B). Previous studies showed that HIF-1α transcriptionally upregulates the expression of MMPs, and in the absence of HIF-1α, the level of MMPs decreased (Du et al., 2008; Palazon et al., 2014). Our findings suggest that NBP promotion of the degradation of HIF-1α may be responsible for the decrease in Mmp-9 levels in RAW 264.7 macrophages.

Figure 6.

NBP inhibits H₂O₂-induced ECM degradation.

(A) RAW 264.7 cells were treated with 200 µM H₂O₂ and 40 µM NBP for 8 hours. NBP inhibited H₂O₂-induced ECM degradation in RAW 264.7 cells. Cells were plated on coverslips coated with FITC-gelatin (green) for 8 hours. F-actin was stained with phalloidin (red), and nuclei were stained with DAPI (blue) (100× magnifications). Black areas indicate gelatin degradation. (B) Mmp-2 and Mmp-9 mRNA levels were detected by real-time polymerase chain reaction. Data normalized by control group are presented as mean ± SD. The experiment was repeated three times. **P < 0.01, vs. control group (one-way analysis of variance followed by Bonferroni correction). CTL: Control; DAPI: 4′,6-diamidino-2-phenylindole; ECM: extracellular matrix; Mmp: matrix metalloproteinase; NBP: Dl-3-n-butylphthalide.

Discussion

Stroke is a common and frequent occurrence in the elderly, often leaves survivors with chronic disability. Stroke is accompanied by cerebral ischemia-reperfusion injury and the death of neurons and other functional cells, leading to motor and cognitive impairment. After acute ischemic stroke, large amounts of ROS are produced, leading to further tissue damage. These ROS damage cellular macromolecules, leading to apoptosis and necrosis. Dysfunction of the BBB, characterized by degradation and increased permeability of the BBB, is an important pathophysiological feature of acute ischemic stroke. NBP has been shown to improve microcirculation and cerebral blood flow; it exhibits anti-inflammatory properties, inhibits apoptosis and protects the vascular endothelium to achieve brain protection (Huang et al., 2018; Wang et al., 2018; Chen et al., 2019; Fan et al., 2021). However, the exact molecular mechanisms of how NBP exerts these effects are unclear.

Most of neuroprotective drugs exert their protective effects by increasing the antioxidant capacity of cells (Tao et al., 2020; Griñán-Ferré et al., 2021). Several studies have shown that NBP exerts a significant neuroprotective effect in cerebral ischemia, which may be from its ability to inhibit oxidative stress (Chen et al., 2018; Que et al., 2021) and reduce brain edema (Mamtilahun et al., 2020; Zeng et al., 2020), neuronal death and inflammation (Liu et al., 2021; Que et al., 2021). Our study showed that NBP exerts a protective effect by affecting the mitochondrial apoptotic pathway, inhibiting Bax and Bnip3 expression. NBP reversed the reduction of mitochondrial membrane potential and activation of caspase 3 caused by H₂O₂. Chromatin immunoprecipitation experiments confirmed that the HIF-1α transcription factor binds the Bax and Bnip3 promoters, and NBP promoted the ubiquitinated-mediated degradation of HIF-1α, thereby inhibiting Bax and Bnip3 expression. HIF-1α protein level is modified by binding to Hippel-Lindau protein (VHL) to form a complex that is subsequently degraded by ubiquitination. We speculate that the effect on the degradation of ubiquitination may be due to the involvement of NBP in the interaction between ubiquitin and HIF-1α, and NBP may contribute to the formation of a more stable binding of the HIF-1α and VHL complex during ubiquitination, thus enhancing the degradation of HIF-1α.

In addition to exhibiting anti-apoptotic and ROS inhibitory effects, NBP treatment also inhibited the degradation of the basement membrane by macrophages, exerting exerting neuroprotective. The underlying mechanism needs further study. Given that NBP affects the ubiquitinated degradation of HIF-1α, which promotes the expression of matrix metalloproteinases, this may be related to the degradation of the basement membrane by macrophages affected by NBP. Further studies are required to clarify the mechanism. In addition, in vivo experiments may further compensate for the limitations of the results of in vitro experiments in this study.

In conclusion, our study provides evidence that NBP induces ubiquitination and degradation of the HIF-1α transcription factor and influences mitochondrial membrane potential and caspase-3 activation, resulting in anti-apoptotic effects and inhibition of ROS-mediated damage. However, how NBP as a small molecule is involved in influencing HIF-1α ubiquitination requires further study. Further studies with more cell types as well as animal experiments may provide further insights. Our study paves the way for further studies on the neuroprotective effects and mechanisms of NBP.

Additional files:

Additional Table 1: List of 31 differentially expressed genes involved in apoptosis.

Additional Table 2: Predicting transcription factors of differentially expressed genes.