Protective effects of brain and muscle ARNT-like gene 1 on oxidized low-density lipoprotein-induced human brain microvascular endothelial cell injury by alleviating ferroptosis

Abstract

Ferroptosis plays an important role in atherosclerotic cerebrovascular diseases. The brain and muscle ARNT-like gene 1 (BMAL1) is an important mediator in the progression of cerebrovascular diseases. However, whether BMAL1 regulates ferroptosis in atherosclerotic cerebrovascular diseases remains obscure. Here, human brain microvascular endothelial cells (HBMECs) were exposed to oxidized low-density lipoprotein (ox-LDL) to imitate cerebrovascular atherosclerosis. It was found that ox-LDL treatment induced ferroptosis events and reduced BMAL1 expression in HBMECs, which could be reversed by ferroptosis inhibitor ferrostatin-1. Furthermore, BMAL1 overexpression markedly mitigated ox-LDL-induced ferroptosis events and cell damage. Moreover, BMAL1 overexpression significantly promoted nuclear factor erythroid 2-related factor 2 (Nrf2) expression in HBMECs under ox-LDL conditions. And, Nrf2 silencing attenuated the protective effects of BMAL1 on ox-LDL-stimulated HBMEC damage and ferroptosis. Altogether, our findings delineate the cerebrovascular protective role of BMAL1/Nrf2 by antagonizing ferroptosis in response to ox-LDL stimulation and provide novel perspectives for therapeutic strategies for atherosclerotic cerebrovascular diseases.

Introduction

Atherosclerosis is a chronic inflammatory vascular disease mediated by innate and adaptive immunity under pathological conditions. As the basic skeleton and key structure of the blood-brain barrier, cerebrovascular endothelial cells are involved in the transport of oxygen and nutrients, and monitor cell metabolism to maintain normal brain function. Many studies have shown that oxidized low-density lipoprotein (ox-LDL)-associated brain microvascular endothelial cell injury is strongly related to the progression of atherosclerotic cerebrovascular diseases.^1,2 Therefore, elucidating the molecular mechanism of ox-LDL-induced brain microvascular endothelial cell injury is of great significance for alleviating cerebrovascular atherosclerosis.

Ferroptosis is a form of programmed cell death, characterized by iron overload and the accumulation of reactive oxygen species (ROS)-dependent lipid peroxides. Numerous studies have demonstrated that ferroptosis is involved in the development and progression of cerebrovascular atherosclerosis via iron, lipid, and amino acid metabolism. ³ Iron is one of the most crucial elements in the human body and iron metabolism imbalance is a common event of cerebrovascular diseases. During the brain microvascular endothelial cell injury period, excess free iron is deposited in cells, ⁴ leading to the Fenton reaction and Haber-Weiss reaction. Fenton or Haber-Weiss reaction catalyzes endogenous hydrogen peroxide (H₂O₂) into highly toxic hydroxyl radical (•OH) through ferrous iron (Fe²⁺), further promoting the production of ROS and lipid peroxidation of unsaturated fatty acids on cell membranes and thus resulting in the occurrence of ferroptosis. ⁵ The downregulated glutathione (GSH) and glutathione peroxidase 4 (GPX4) is also a major feature of ferroptosis. ⁶ Yasir Abdul and his colleagues ⁷ demonstrated that deferoxamine treatment alleviated increased blood-brain barrier permeability and neurovascular remodeling after stroke in diabetic rats. Further experiments verified that ferroptosis markers and ROS could be triggered by iron in cerebrovascular endothelial cells from diabetic animals. It was reported that ferroptosis was also involved in oxygen and glucose-deprivation-induced brain microvascular endothelial cell dysfunction. ⁸

Brain and muscle ARNT-like gene 1 (BMAL1) protein is an important member of molecular circadian oscillators in mammals. ⁹ Numerous studies have shown that BMAL1 also participates in a variety of pathological processes, such as oxidative stress, inflammatory response, and blood-brain barrier integrity. A study of an intracerebral hemorrhage (ICH) rat model by injection of autologous blood reported that the expression of BMAL1 was significantly reduced in ICH rat brain, while BMAL1 overexpression alleviated ICH-induced oxidative stress, inflammation, brain edema, blood-brain barrier damage, neuronal death, and neurological dysfunction. ¹⁰ Furthermore, BMAL1 expression was also decreased in the cerebral cortex of traumatic brain injury rats, especially at 48 h, and the recombinant BMAL1 protein was found to reduce brain edema, neurobehavioral injury, somatosensory impairment, nerve cell necrosis, and apoptosis. ¹¹ These studies confirmed the important protective role of BMAL1 in cerebrovascular diseases. However, the role of BMAL1 in ox-LDL-induced ferroptosis in brain microvascular endothelial cells remains unclear. In the current research, we unveiled the effect of BMAL1 on ox-LDL-triggered ferroptosis and cell damage in human brain microvascular endothelial cells (HBMECs) as well as the possible mechanisms involved, to broaden our understanding of the endothelial injury.

Materials and methods

Cell culture and treatment

HBMECs were obtained from Procell (Wuhan, China) and cultured in a complete culture medium (CM-H124, Procell) at a 37°C incubator with 5% CO₂.

For ox-LDL and ferrostatin-1 treatment, HBMECs were pretreated ferrostatin-1 (HY-100579, MCE, USA) for 30 min and then treated with 100 mg/L ox-LDL (Solarbio, Beijing, China) for 24 h according to a previous study. ¹²

Cell transfection

BMAL1-expressing plasmids (BMAL1_OE), small interference RNA for Nrf2 (si-Nrf2), or their negative controls (vector and si-NC) were obtained by GenePharma (Shanghai, China). These plasmids were transfected into HBMECs using Lipofectamine 3000 (ThermoFisher, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNAs were isolated from HBMECs using TRIzol reagent (Invitrogen, CA, USA). The synthesis of cDNA used PrimeScript RT-PCR Kit (Takara, Dalian, China). QRT-PCR was performed using SYBR Green PCR Master Mix (Invitrogen, USA). The relative abundance of BMAL1 and Nrf2 was calculated by the 2^−ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) acts as the internal reference. The following primer sequences were used: BMAL1-F: 5′-ATG​TGG​AAT​CCT​GGG​CCT​TC-3′, BMAL1-R: 5′-TTT​CAG​GCG​GTC​AGC​TTC​TT-3′; Nrf2-F: 5′-AGG​TTG​CCC​ACA​TTC​CCA​AA-3′, Nrf2-R: 5′-ACG​TAG​CCG​AAG​AAA​CCT​CA-3′.

Western blot analysis

RIPA Lysis Buffer (Beyotime, Shanghai, China) was used for cell lysis. BCA Protein Assay Kit (Beyotime) was used for protein quantification. Protein was separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane (Millipore, USA). After blocking with 5% skim milk, the membrane was incubated with primary antibody overnight at 4°C on a shaker. Then, the membrane was incubated with secondary antibody for 1 h. The protein bands were developed with Western Chemiluminescent HRP Substrate (Millipore, USA). Anti-BMAL1 (ab3350), anti-TFR1 (ab10579), anti-Nrf2 (ab137550), and anti-GAPDH (ab9485) antibodies were purchased from Abcam (USA). Anti-ACSL4, anti-GPX4 anti-FTH1 antibodies were obtained from Proteintech (Wuhan, China),

Cell counting kit 8 (CCK-8)

HBMECs were cultured in a 96-well plate (5 × 10⁴ cells/well). After indicated treatments, cells were incubated with CCK-8 reagent (Solarbio, Beijing, China) for 1 h. Absorbance at 450 nm was detected using a microplate reader.

5-Ethynyl-2′-deoxyuridine (EdU) assay

HBMECs were stained with EdU solution (RiboBio, Guangzhou, China) for 2  h. Then, cells were incubated with 4′,6-diamidino-2-phenylindole (DAPI). Cells were analyzed under fluorescence microscopy.

Tube formation assay

In Vitro Angiogenesis Assay Kit (Sigma-Aldrich, USA) was adopted for evaluation of tube formation by endothelial cells as previously described. ¹³ Briefly, 50 μL of diluted ECMatrix solution was transferred into each well of the 96-well tissue culture plate and incubated at 37°C for 1.5 h. HBMECs were seeded onto the surface of the polymerized ECMatrix and incubated at 37°C for 16 h. Tube formation was inspected under an inverted light microscope.

Flow cytometry

Annexin V-FITC Apoptosis Kit (Vazyme, Nanjing, China) was utilized for assessing cell apoptosis. HBMECs were incubated with Annexin V-FITC and PI for 15 min. Cells were measured using the FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, USA).

Measurement of intracellular ROS levels

Intracellular ROS levels were examined with Fluorometric Intracellular ROS Kit (Solarbio, Beijing, China). DCFH-DA fluorescent probe was used for ROS detection. DCFH-DA itself has no fluorescence and can freely cross the cell membrane. After entering the cell, DCFH can be hydrolyzed to produce DCFH. Non-fluorescent DCFH can be oxidized to produce fluorescent DCF by intracellular ROS. DCF fluorescence intensity indicates the levels of intracellular ROS. In brief, DCFH-DA was diluted with a serum-free medium to 10 μmol/L. Cells were collected and suspended in diluted DCFH-DA and incubated at 37°C for 20 min. The cells were washed three times with serum-free medium and then examined by fluorescence microscopy or flow cytometry.

Iron assay

Iron Assay Kit (Abcam) was used to measure iron concentration following the kit’s instructions. Free ferrous iron (Fe²⁺) reacts with Iron Probe to produce a stable colored complex with absorbance at 593 nm. Samples and standards were added into each well, and incubated in assay buffer for 30 min at 37°C. The sample was added with an iron probe and incubated for 60 min at 37°C. The analysis was carried out with a microplate reader.

Measurement of GSH activity

GSH Assay Kit (Jiancheng, Nanjing, China) was used to measure cellular GSH levels following the kit’s instructions. Determination principle: The complex formed by the reaction of DTNB and GSH has a characteristic absorption peak at 412 nm, and its absorption value is proportional to the content of GSH. After cells were washed with PBS, the precipitated cells were collected by centrifugation at low speed, resuspended in isotonic PBS buffer, and then crushed by ultrasonic grinding. After adding corresponding reagents into blank, standard, and determination wells according to the instructions, cells were incubated for 5 min. The absorbance was measured at 405 nm by a microplate reader.

Statistical analysis

GraphPad Prism 8.0 was used for statistical analysis. Data were shown as the mean ± standard deviation (SD) from three independent experiments. Multiple sets of data were compared using one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. p < 0.05 was considered significant.

Results

Ox-LDL treatment induces ferroptosis in HBMECs

To clarify whether ferroptosis is involved in the ox-LDL-induced HBMEC injury, the ferroptosis inhibitor ferrostatin-1 (5 μm) was used to treat ox-LDL-exposed HBMECs. As displayed in Figure 1(a) and (b), ox-LDL was observed to inhibit cell survival and proliferation. However, ferrostatin-1 reversed this inhibitiory effect (p < 0.01). Meantime, ox-LDL treatment also changed ferroptosis-related markers, including increased ROS level, Fe²⁺ content, and reduced GSH activity. However, ferrostatin-1 could reverse these changes (Figure 1(c)–(e)). Additionally, we also analyzed the expression change of Acyl-CoA synthetase long-chain family member 4 (ACSL4) and GPX4, ferritin heavy chain 1 (FTH1), and transferrin receptor 1 (TFR1), which are major regulators of ferroptosis. As displayed in Figure 1(f), ox-LDL remarkably elevated ACSL4 and TFR1 (two positive regulators) levels, and decreased GPX4 and FTH1 (two negative regulators) levels in HBMECs. Dramatically, ferrostatin-1 reversed the change of these proteins (Figure 1(f)). These findings reveal that ferroptosis is involved in ox-LDL-induced HBMEC damage.

Figure 1.

Ox-LDL promotes HBMEC ferroptosis. After HBMECs were treated with ox-LDL alone or in combination with ferrostatin-1 (1 μm) for 24 h, (a) Cell survival was assessed by CCK-8 assay. (b) Cell proliferation was detected by EdU. (c) Iron concentration was detected. (d) Intracellular ROS levels were measured. (e) Quantification of cellular MDA levels. (f) Quantification of cellular GSH levels using the DTNB method. (g) Western blot was performed to measure the expression of ACSL4, GPX4, FTH1, and TFR1. Fer, ferrostatin-1. **p < 0.01, ***p < 0.001 vs. the Control group; ##p < 0.01, ###p < 0.001 vs. the ox-LDL group.

BMAL1 inhibits ox-LDL-incubated HBMEC damage

Firstly, we found that ox-LDL exposure significantly reduced BMAL1 gene and protein expression, compared with the Control group, but ferrostatin-1 could reverse these changes (Figure 2(a) and (b)). To figure out the role of BMAL1 in ox-LDL-stimulated HBMEC damage, BMAL1-expressing plasmids (BMAL1_OE) were transfected into HBMECs. The results showed that BMAL1-expressing plasmids also significantly elevated BMAL1 under ox-LDL conditions (p < 0.01, Figure 2(c) and (d)). Moreover, to explore the effect of BMAL1 overexpression on HBMEC proliferation, cell viability, and EdU assays were performed following BMAL1-expressing plasmids transfection and ox-LDL treatment. It was found that BMAL1 overexpression promoted cell proliferation in HBMECs (p < 0.05, Figure 2(e) and (f)). Moreover, the flow cytometry results indicated that ox-LDL induced the apoptosis of HBMECs (26.13 ± 0.95% in the ox-LDL group vs 3.92 ± 0.11% in the NC group). Similarly, BMAL1 overexpression remarkably alleviated apoptosis triggered by ox-LDL exposure (11.31 ± 0.63% in ox-LDL + BMAL1_OE group vs 3.92 ± 0.11% in ox-LDL + NC group, p < 0.05, Figure 2(g)). In addition, BMAL1 overexpression could promote tube formation in HBMEC cells under ox-LDL conditions (Figure 2(h)).

Figure 2.

BMAL1 protected against ox-LDL-incubated HBMEC damage. HBMECs were transfected with BMAL1-expressing plasmids, followed by incubation with ox-LDL. (a) BMAL1 mRNA levels were quantified using qRT-PCR. (b) BMAL1 protein expression was measured by Western blot. (c) BMAL1 mRNA expression was examined by qRT-PCR. (d) BMAL1 protein expression was analyzed by Western blot. (e) The cell survival rate is determined by CCK-8. (f) Cell proliferation was detected by EdU. (g) Cell apoptosis was determined by Annexin V-FITC staining followed by flow cytometric analysis. Representative images (left) and quantification (right) of HBMECs apoptosis. (h) Representative images (left) and quantification (right) of HBMECs tube formation. **p < 0.01, ***p < 0.001 vs. the Control group; ##p < 0.01, ###p < 0.001 vs. ox-LDL group.

BMAL1 protectes HBMECs against ox-LDL-induced ferroptosis

To uncover the role of BMAL1 in ferroptosis, cellular iron concentration, lipid ROS, and GSH were measured after BMAL1-expressing plasmids transfection and ox-LDL treatment. As shown in Figure 3(a)–(c), ox-LDL treatment increased intracelluler iron amount and ROS levels, and significantly decreased GSH activity. However, these effects were all reversed by BMAL1 overexpression. Additionally, we also analyzed the expression of ACSL4, GPX4, FTH1, and TFR1. As displayed in Figure 3(d), ox-LDL remarkably elevated ACSL4 and TFR1 levels, and decreased GPX4 and FTH1 levels in HBMECs. Dramatically, BMAL1 overexpression reversed the change of these proteins. These findings reveal that the BMAL1 resists ox-LDL-stimulated ferroptosis.

Figure 3.

BMAL1 protected HBMECs against ox-LDL-induced ferroptosis. HBMECs were transfected with BMAL1-expressing plasmids, followed by incubation with ox-LDL. (a) Intracellular ROS levels were measured. (b) Intracellular iron levels were detected. (c) Cellular GSH activity was detected. (d) Western blot was performed to assess the expression of ACSL4, GPX4, FTH1, and TFR1 proteins. *p < 0.05, ***p < 0.001 vs. the Control group; #p < 0.05, ##p < 0.01 vs. the ox-LDL group.

BMAL1 inhibits ox-LDL-induced HBMEC injury and ferroptosis by modulating Nrf2

BMAL1 has been reported to activate Nrf2 signaling, which is a key intracellular antioxidant pathway. Here, we found that BMAL1 overexpression notably restored the expression of the Nrf2 gene (Figure 4(a)) and protein expression (Figure 4(b)) under the ox-LDL condition, indicating that Nrf2 may be a downstream target of BMAL1. To further confirm whether BMAL1 regulates ox-LDL-induced damage through Nrf2, HBMECs were co-transfected with BMAL1-expressing plasmids and si-Nrf2. As summarized in Figure 4(c), the efficiency of Nrf2 knockdown was verified. Depletion of Nrf2 partly dismissed the anti-ferroptosis function of BMAL1 overexpression, evidenced by increased ROS and the iron amount and decreased GSH content (Figure 4(d)–(f)).

Figure 4.

BMAL1 inhibited ox-LDL-induced ferroptosis of HBMECs through modulating Nrf2. After HBMECs were transfected with BMAL1-expressing plasmids, followed by incubation with ox-LDL, (a) Nrf2 mRNA expression was examined by qRT-PCR, and (b) Nrf2 protein expression was analyzed by Western blot. **p < 0.01 vs. the Control group; ##p < 0.01 vs. the ox-LDL group. (c) Western blot was conducted to measure Nrf2 protein levels in HBMECs transfected with si-Nrf2. **p < 0.01 vs. the si-NC group. After HBMECs were co-transfected with BMAL1-expressing plasmids and si-Nrf2 or alone, followed by incubation with ox-LDL, (d) intracellular ROS, (e) iron levels, and (f) GSH activity were analyzed. #p < 0.05 vs. the ox-LDL group. &p < 0.05 vs. the ox-LDL + BMAL1_OE group.

Besides this, the transfection of si-Nrf2 also inhibited viability, as evidenced by CCK8 and EdU assays (Figure 5(a) and (b)). Furthermore, BMAL1 overexpression induced cell apoptosis, which was abolished by Nrf2 depletion in HBMECs under ox-LDL conditions (Figure 5(c)). Finally, Nrf2 knockdown also inhibited tube formation rate, compared to that of HBMECs transfected with BMAL1_OE alone (Figure 5(d)). These findings suggest that BMAL1 upregulates Nrf2 to protect HBMECs against ox-LDL-induced ferroptosis and cell injury.

Figure 5.

BMAL1 inhibited ox-LDL-induced HBMEC injury by modulating Nrf2. HBMECs were co-transfected with BMAL1-expressing plasmids and si-Nrf2 or alone, followed by incubation with ox-LDL. (a) The cell survival rate is determined by CCK-8. (b) Cell proliferation was detected by EdU staining. (c) Cell apoptosis was determined by Annexin V-FITC staining followed by flow cytometric analysis. Representative images (left) and quantification (right) of HBMECs apoptosis. (d) Representative images (left) and quantification (right) of HBMECs tube formation. #p < 0.05 vs. the ox-LDL group. &p < 0.05 vs. the ox-LDL + BMAL1_OE group.

Discussion

Atherosclerosis is a chronic inflammatory disease of the vascular system and is the leading cause of cerebrovascular diseases. ¹⁴ Endothelial dysfunction caused by oxidative lipid deposition is the main cause of early atherosclerosis. Therefore, elucidating the mechanism of endothelial dysfunction induced by oxidative lipid deposition may provide promising intervention targets for the treatment of atherosclerosis Herein, we found that ox-LDL-induced HBMEC damage, ferroptosis, and reduced BMAL1 expression in HBMECs, which were all reversed by ferrostatin-1 treatment. Furthermore, overexpression of BMAL1 protected HBMECs against ox-LDL-induced damage and ferroptosis. Mechanistically, BMAL1 upregulated the expression of Nrf2 to protect HBMECs against ox-LDL. This study clarifies the protective effect of BMAL1 on ox-LDL-induced injury by resisting ferroptosis, providing a novel perspective for the prevention and treatment of atherosclerosis.

Brain microvascular endothelial cells are a major component of the blood-brain barrier and maintain the homeostasis of the cerebrovascular system. Vascular endothelial cells constitute the endothelial surface of blood vessels, acting as a structural barrier to avoid all kinds of poison to tissue damage and inflammatory damage. Recent studies have confirmed the involvement of ferroptosis in the pathogenesis of cerebrovascular diseases and endothelial dysfunction. ¹⁵ Tuo et al. ¹⁶ provided evidence of the occurrence of ferroptosis events in vitro diabetic brain ischemic injury model. Chen C et al. ⁸ reported that ferroptosis occurred in BMECs of ICH models in vitro. Furthermore, deferoxamine treatment prevents post-stroke vasoregression and improves BBB permeability in diabetic rats., and prevented the increase in ferroptosis-related markers and lipid ROS in iron-induced BMECs by iron chelation. ⁷ Gao S and his colleagues ¹⁷ demonstrated that endothelial cells and microglia undergo ferroptosis in a SAH model. In this present study, we revealed that HBMECs undergo ferroptosis after ox-LDL treatment in vitro. Interestingly, ferrostatin-1 could reverse ox-LDL-induced ferroptosis and cell damage, indicating the involvement of ferroptosis in endothelial dysfunction.

Crucially, our study identified that BMAL1 is an important regulator in ox-LDL-induced HBMEC damage. Previous studies have demonstrated the regulatory role of BMAL1 in endothelial homeostasis. Loss of BMAL1 in mouse endothelial cells increases chemokine expression, impairs endothelial integrity and barrier function, and subsequently increases leukocyte transport across the endothelial layer. ¹⁸ BMAL1 inhibited ox-LDL-stimulated ROS generation and subsequent endothelial-to-mesenchymal transition in human aortic endothelial cells. ¹⁹ Our results also revealed that BMAL1 overexpression reduced the intracellular iron concentration and lipid ROS generation and increased GSH levels in HBMECs under ox-LDL conditions.

BMAL1 has recently been reported to regulate Nrf2 and Nrf2-mediated antioxidant pathways.^20,21 BMAL1 has been shown to activate Nrf2-mediated antioxidant pathways in macrophages, thereby reducing interleukin-1beta production. Consistent with these findings, we confirmed that overexpression of BMAL1 up-regulated Nrf2 expression in this study. Nrf2 has been reported to regulate the transcription of lipid peroxidation-related genes, thereby participating in ferroptosis.^22,23 As an example, genetic or pharmacological upregulation of Nrf2 suppressed ferroptosis and mitochondrial dysfunction in neurodegeneration, implying the central role of Nrf2 in ferroptosis. ²⁴ Here, rescue experiments demonstrated that depletion of Nrf2 abolished the protective effect of BMAL1 on cell injury in HBMECs. These results suggested that BMAL1 protected against ferroptosis-mediated HBMEC damage by activating Nrf2 signaling.

Conclusions

In summary, we present evidence that BMAL1 exerts a protective effect on ox-LDL-mediated HBMEC damage and ferroptosis. Mechanically, BMAL1 treatment upregulats the expression of Nrf2, and as a result, alleviats ferroptosis-mediated HBMEC damage. This study reveal that BMAL1 might be a promising target for the treatment of atherosclerotic cerebrovascular diseases.