Protective effects of ginsenoside Rb1 on H2O2-induced oxidative injury in human endothelial cell line (EA.hy926) via miR-210.

Ginsenoside Rb1 (Rb1) possesses a cardioprotective effect via mediating microRNAs (miRs), while it is unexplored whether miR-210 is regulated by Rb1 in response to oxidative stress. Human endothelial EA.hy926 cells were stimulated with H2O2 before Rb1 treatment. After transfection, cell viability, apoptosis, migration, and invasion assays were conducted. Western blot was applied to quantify protein. BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) and miR-210 were analyzed with quantitative reverse transcription polymerase chain reaction. Dual luciferase activity assay was performed. Rb1 elevated viability, migration, and invasion of H2O2-treated cells. H2O2-induced apoptosis was moderated by Rb1. miR-210 was augmented in H2O2-treated cells after Rb1 stimulation. miR-210 inhibitor abolished the positive effects of Rb1. BNIP3 was negatively modulated by miR-210 and implicated in modulating viability, apoptosis, and migration and invasion. In addition, BNIP3 modulated phosphorylation of regulators. Rb1 repressed oxidative injury via elevating miR-210. miR-210 negatively mediated BNIP3, which participated in oxidative damage via regulating mammalian targets of rapamycin (mTOR) and nuclear factor-κB (NF-κB).

Introduction

The pathogenesis of neurodegenerative diseases is directly associated with the damage of endothelial cells, which serve as a target for reactive oxygen species (ROS) and consequently are vulnerable to oxidative damage.[1] Through causing the dysfunction of antioxidant proteins and pro-oxidant enzymes, oxidative stress perturbs mitochondrial properties and subsequently triggers the cleaved progress of caspase-3 and caspase-9 via oxidative stress-mediated signaling pathways.[2] Once the endothelial cell death is initiated, the endothelial barrier feature is gravely disrupted and the cerebrovascular diseases are therewith contributed since it plays a pivotal role in the development of neurological function.[3] Numerous pharmaceutical studies have been performed to mitigate the cerebral vascular damage based on account of part antioxidant capability.[4,5]

Ginsenoside Rb1 (Rb1), a steroidal saponin compound, is the main bioactive component of ginseng. Extensive evidence have demonstrated that Rb1 alleviates oxidative stress-induced apoptosis and improves cell viability under the stressful environments.[6,7] A previous study has shown that Rb1 substantially maintains the redox homeostasis by scavenging hydroxyl radical and hypochlorous acid.[8] A recent study presented a microRNA (miR)-relative mechanism, whereby Rb1 exhibits a cardioprotective effect on hypoxic- and ischemic-damaged cardiomyocytes.[9] However, it still remains indistinct whether miR-210 is associated with the protective function of Rb1 against oxidative stress-mediated endothelial cell injury.

miR-210 is regulated by hypoxia inducible factor (HIF) in response to hypoxia stimulus.[10] The function of miR-210 has been studied in several physiopathological conditions. A foregone study highlighted that lung adenocarcinoma cells overexpressing miR-210 suggest a specific appearance characterized by abundance of transcripts associated with cell death and mitochondrial dysfunction.[11] As for its function in the post-injury neurogenesis, miR-210 participates in mediating the mitochondrial function and modulating apoptosis.[12] The role of miR-210 in cell viability and apoptosis has been elaborated under oxidative stress, which exhibits an anti-oxidative property.[13] BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3), as a crucial mitochondrial redox sensor that mediates cell death in response to oxidative stress,[14] has been reported to be directly modulated by miR-210 to reduce the hypoxia-induced cell death,[15] whereas its downstream cascades are still unexplored in endothelial cells under oxidative stress.

As a consequence, this study was aimed to investigate the cytoprotective function of Rb1 in the H2O2-induced oxidative damaged cell model. In addition, we explored the underlying mechanism which might be associated with the miR-210. Considering that BNIP3 is an essential redox sensor, we further studied its modulatory effects on cell viability, migration, invasion and apoptosis as well as the correlative signaling pathways.

Materials and methods

Cell culture and treatment

The human endothelial EA.hy926 cells were derived from American Type Culture Collection (ATCC, Rockville, MD, USA). EA.hy926 cells were cultured in high glucose-Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St. Louis, MO, USA) containing 10% fetal calf serum (FBS; Gibco, Gaithersburg, MD, USA), 5 mM hypoxanthine (Sigma), 0.02 mM aminopterin (Sigma), and 0.8 mM thymidine (Sigma), in a humidified incubator containing 95% air and 5% CO2 at 37°C. EA.hy926 cells were stimulated with 150 μM H2O2 (Sigma) for 2 h. Before stimulation with H2O2, EA.hy926 cells were pre-incubated with 20 μM Rb1 for 1 h. Rb1 was purchased from Sigma.

Cell viability assay

EA.hy926 cells (1 × 105 cells/well) were plated in 24-well plates. After stimulation with H2O2 and/or pre-incubation with Rb1, EA.hy926 cells were trypsinized and stained with trypan blue solution (Gibco) and counted with cell-counting chamber under a microscope.

Apoptosis assay

A flow cytometry in combination with Annexin V–fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) apoptosis detection kit (Biosea, Beijing, China) were applied to detect apoptotic cells. Briefly, EA.hy926 cells were seeded in six-well plates at a density of 1 × 105 cells/well. After stimulation with H2O2 and/or pre-incubation with Rb1, EA.hy926 cells were washed twice with pre-cold phosphate-buffered saline (PBS; Sigma). After staining with Annexin V-FITC/PI, apoptotic cells were distinguished with a flow cytometer (Beckman Coulter, Brea, CA, USA).

Migration and invasion assay

Cell migration activity was examined with a modified Boyden chamber with 8 μm pores. EA.hy926 cells were supplemented with 200 μL serum-free medium and seeded on the upper compartment. The lower compartment was supplemented with 600 μL complete medium. H2O2 or Rb1 was added to both inserts and wells. Cells were fixed with methanol after incubation at 37°C for 8 h. Traversed cells on the lower side of the filter were stained with crystal violet and counted. The invasive behavior of EA.hy926 cells was determined with a BD BioCoatTM MatrigelTM invasion (BD Biosciences, San Jose, CA, USA). Serum-free medium was supplemented into the inserts and the wells. EA.hy926 cells were plated onto the inserts. H2O2 or Rb1 was added to both inserts and wells. Chambers were incubated for 24 h at 37°C. After fixation with methanol and staining with crystal violet, the invasive cells were counted.

Transfection

miR-210 mimic, miR-210 inhibitor, scramble, and negative control (NC) were provided by GenePharma (Shanghai, China). Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) was employed to transfect miR-210 mimic, miR-210 inhibitor, scramble, and NC into EA.hy926 cells by following manufacturer’s instructions.

Full-length BNIP3 sequence and short hairpin (sh)-BNIP3 directly against BNIP3 were ligated into pEX-3 and pGU6/Neo plasmids (GenePharma) named pEX-BNIP3 and sh-BNIP3, respectively. The empty plasmids served as NCs named pEX and sh-NC. After transfection using lipofectamine 3000, the stably transfected cells cultured in the medium containing 0.5 mg/mL G418 (Sigma) for selecting the G418-resistant cell clones.

Dual luciferase activity assay

Fragments of the BNIP3 3′-untranslated region (UTR) containing the putative miR-210 binding site (BNIP3 promoter) or U6 (U6) were generated by polymerase chain reaction (PCR). Amplified products were ligated into the pMIR-report vector (Life Technologies, Carlsbad, CA, USA). EA.hy926 cells were cotransfected with miR-210 mimic or NC and BNIP3 promoter or U6. Renilla luciferase reporter acted as an internal control. The dual-luciferase assay system (Promega, Madison, USA) was used to measure the luciferase activities after transfection for 24 h according to manufacturer’s instructions.

Quantitative reverse transcription PCR

Total RNA was extracted from EA.hy926 cells with Trizol reagent (Life Technologies). The expression of miR-210 was quantified with TaqMan MicroRNA Reverse Transcription Kit and TaqMan Universal Master Mix II with the TaqMan MicroRNA Assay of miR-210 and U6 (Applied Biosystems, Foster City, CA, USA). U6 served as an internal reference. The mRNA expression of BNIP3 was determined using RNA-to-cDNA Master Mix and TaqMan primers (Applied Biosystems). Its relative expression was normalized with an endogenous control Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) using the Ct method.

Western blot

The total proteins were quantified with the BCATM protein assay kit (Pierce, Appleton, WI, USA) after extracting using radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology, Shanghai, China). The Bio-Rad Bis-Tris Gel system was established for Western blot assay according to manufacturer’s protocol. GAPDH served as an internal reference. After separation, proteins were electrophoretically transferred onto polyvinylidene difluoride (PVDF) membrane (Invitrogen). Membranes were blocked with 5% bovine serum albumin (BSA; Sigma). Primary antibodies against proteins were used, including Bcl-2 (ab32124; 1:1000), Bax (ab53154; 1:1000), pro caspase-3 (ab32499; 10,000), active caspase-3 (ab32042; 1:500), pro caspase-9 (ab138412; 1:1000), active caspase-9 (ab32042; 1:500), BNIP3 (44060; 1:1000) (all purchased from Abcam, Cambridge, UK), S6 (2217; 1:1000), phospho (p)-S6 (4858; 1:2000), mammalian targets of rapamycin (mTOR) (2983; 1:1000), p-mTOR (5536; 1:1000), inhibitor of nuclear factor κ-B α (IκBα) (4812; 1:1000), p-IκBα (2859; 1:1000), p65 (8242; 1:1000), p-p65 (3033; 1:1000), and GAPDH (5174; 1:1000) (all purchased from Cell Signaling Technology, Danvers, MA, USA). After incubation with primary antibodies for overnight, the PVDF membrane was incubated with a secondary antibody (7074) (Cell Signaling Technology) for 1 h at room temperature. Chemiluminescence was excited by 200 μL Immobilon Western Chemiluminescent horseradish peroxidase (HRP) substrate (Millipore, Billerica, MA, USA). The protein signals were captured by Image LabTM software (Bio-Rad, Shanghai, China).

Statistical analysis

All the experiments were repeated at least for three times. Data were expressed as mean ± standard deviation (SD). The comparison of all results was conducted with Student’s t-test and one-way analysis of variance (ANOVA). The statistical significance was considered when P was less than 0.05.

Results

Rb1-protected EA.hy926 cells against H2O2-induced damage

In order to investigate whether Rb1 protected EA.hy926 cells against H2O2-induced damage, we established an oxidative damaged cell model. EA.hy926 cells were overtly injured after treatment with 150 μM H2O2 for 2 h, demonstrated by reduction in cell viability (P < 0.01, Figure 1(a)), migration (P < 0.05, Figure 1(b)), invasion (P < 0.05, Figure 1(c)), and induction of apoptotic cells (P < 0.001, Figure 1(d)) as well as aberrant expression of apoptosis-related proteins (Figure 1(e)). Subsequently, we pre-treated EA.hy926 cells with 20 μM Rb1 for 1 h. In contrast to cells in the H2O2 group, more EA.hy926 cells pre-treated with Rb1 survived after being exposed to H2O2 (P < 0.05, Figure 2 (a)). As for migration and invasion, the distinct induction was achieved in H2O2-induced EA.hy926 cells pre-incubated with Rb1 in comparison with the H2O2 group (P < 0.05, Figure 2(b) and (c)). A marked inhibition of apoptosis was observed in H2O2-induced EA.hy926 cells by Rb1 (P < 0.05, Figure 2(d)). Furthermore, Rb1 upregulated Bcl-2 protein expression, repressed Bax protein expression and the production of active caspase-3 and active caspase-9 in H2O2-induced EA.hy926 cells (Figure 2(e)). Collectively, these results suggested that Rb1 protected EA.hy926 cells against H2O2-inudced oxidative injury.

Figure 1.

Oxidative injury was induced with H2O2 in EA.hy926 cells: (a) cell viability was detected after staining with trypan blue. (b) Relative migration was assessed by a modified Boyden chamber. (c) A BD BioCoat Matrigel invasion was performed to analyze the invasive behavior. (d) Apoptotic cells were observed with flow cytometry after staining with Annexin V-FITC/PI. (e) Protein levels were examined with Western blot assay. EA.hy926 cells were treated with 150 μM H2O2 for 2 h.

Annexin V-FITC/PI: Annexin V–fluorescein isothiocyanate/propidium iodide.

*P < 0.05, **P < 0.01, or ***P < 0.001 compared to control cells. n = 3–5.

Figure 2.

H2O2-mediated oxidative injury was repressed by Rb1: (a) cell viability was detected after staining with trypan blue. (b) Relative migration was assessed by a modified Boyden chamber. (c) A BD BioCoat Matrigel invasion was performed to analyze the invasive behavior. (d) Apoptotic cells were observed with flow cytometry after staining with Annexin V-FITC/PI. (e) Protein levels were examined with Western blot assay. EA.hy926 cells were treated with 150 μM H2O2 for 2 h after pre-incubation with/without 20 μM Rb1 for 1 h.

Annexin V-FITC/PI: Annexin V–fluorescein isothiocyanate/propidium iodide; Rb1: ginsenoside Rb1.

*P < 0.05, **P < 0.01, or ***P < 0.001 compared to control cells; #P < 0.05 compared to H2O2 cells. n = 3–5.

Rb1 repressed oxidative damage induced by H2O2 via upregulating miR-210

A recent research revealed that ROS generation induces miR-210 expression, which mediates proliferation and migration.[16] We found that miR-210 expression was evidently augmented in H2O2-induced cells after stimulation with Rb1 (P < 0.01), although it was not significantly mediated by H2O2 (P > 0.05, Figure 3(a)). For confirming that Rb1 protected EA.hy926 cells against oxidative injury through upregulating miR-210, we silenced miR-210 by transfecting miR-210 inhibitor into EA.hy926 cells. The transfection efficiency was notable. As shown in Figure 3(b), miR-210 was upregulated by miR-210 mimc (P < 0.01) while downregulated by miR-210 inhibitor (P < 0.01). Our results indicated that the increment in cell viability, migration, and invasion along with the decrement in apoptotic cells by Rb1 were prominently attenuated by the transfection of miR-210 inhibitor in H2O2-induced EA.hy926 cells (P < 0.05 or P < 0.01, Figure 4(a)–(d)). In addition, miR-210 silence facilitated apoptosis evidenced by lessened Bcl-2, enhancive Bax, as well as activated caspase-3 and caspase-9 (Figure 4(e)). Therefore, we concluded that Rb1 protected EA.hy926 cells against oxidative damage induced by H2O2 via upregulating miR-210.

Figure 3.

Rb1 elevated miR-210 expression: (a) miR-210 expression was quantified in EA.hy926 cells after stimulation with 150 μM H2O2 for 2 h after pre-incubation with/without 20 μM Rb1 for 1 h. (b) Profile of miR-210 expression was quantified in EA.hy926 cells after transfection with miR-210 mimic, miR-210 inhibitor, scramble, or NC. miR-210 was analyzed with qRT-PCR.

miR-210: microRNA-210; Rb1: ginsenoside Rb1; NC: negative control; ns: not significant; qRT-PCR: quantitative reverse transcription polymerase chain reaction.

ns compared to control cells; ##P < 0.01 compared to H2O2 cells; ++P < 0.01 compared to NC cells. n = 3–5.

Figure 4.

miR-210 inhibitor abrogated the protective effects of Rb1 against H2O2-induced oxidative damage: (a) cell viability was detected after staining with trypan blue. (b) Relative migration was assessed by a modified Boyden chamber. (c) A BD BioCoat Matrigel invasion was performed to analyze the invasive behavior. (d) Apoptotic cells were observed with flow cytometry after staining with Annexin V-FITC/PI. (e) Protein levels were examined with Western blot assay. EA.hy926 cells were treated with 150 μM H2O2 for 2 h and/or pre-incubated with/without 20 μM Rb1 for 1 h after transfection.

Annexin V-FITC/PI: Annexin V–fluorescein isothiocyanate/propidium iodide; Rb1: ginsenoside Rb1; miR-210: microRNA-210; NC: negative control.

*P < 0.05, **P < 0.01, or ***P < 0.001 compared to control cells; #P < 0.05 or ##P < 0.01 compared to H2O2 cells; and +P < 0.05 or ++P < 0.01 compared to H2O2 + Rb1 + NC cells. n = 3–5.

miR-210 negatively regulated BNIP3 expression by targeting BNIP3 3′-UTR

BNIP3 has been verified as a redox sensor where increased oxidative stress induces homodimerization and activation of BNIP3.[14] To investigate the role of miR-210, we artificially promoted and inhibited the expression of miR-210 as shown in Figure 3(b) (both P < 0.01). Our results illustrated that miR-210 negatively regulated BNIP3 expression at mRNA and protein levels (P < 0.05 or P < 0.01, Figure 5(a)). In addition, our results from dual luciferase activity assay implied that miR-210 mediated BNIP3 expression by targeting BNIP3 3′-UTR (Figure 5(b)).

Figure 5.

miR-210 negatively mediated BNIP3 expression by targeting BNIP3 3′-UTR: (a) relative mRNA expression of BNIP3 was quantified with qRT-PCR assay. BNIP3 protein level was evaluated with Western blot method. (b) EA.hy926 cells were co-transfected with miR-210 mimic or NC along with reporter constructs containing BNIP3 promoter or U6. Renilla luciferase reporter acted as an internal control. Relative luciferase activity was normalized with Renilla luciferase activity.

3′-UTR: 3′-untranslated region; BNIP3: BCL2/adenovirus E1B 19-kDa interacting protein 3; miR-210: microRNA-210; NC: negative control; ns: not significant; qRT-PCR: quantitative reverse transcription polymerase chain reaction.

ns compared to control cells; #P < 0.05 compared to scramble cells; ++P < 0.01 compared to NC cells; and *P < 0.05 compared to NC cells. n = 3–5.

H2O2 induced oxidative damage via upregulating BNIP3

To confirm that BNIP3 participated in H2O2-induced oxidative injury, we effectually promoted or repressed BNIP3 expression by transfecting pEX-BNIP3 or sh-BNIP3 into EA.hy926 cells, respectively (P < 0.01, Figure 6(a)). Our results showed that BNIP3 overexpression further promoted H2O2-induced decrease in cell viability, migration, invasion, and increment of apoptosis (P < 0.05 or P < 0.01, Figure 6(b)–(f)). However, BNIP3 silence reversed the negative effects of H2O2 on cell viability, migration, invasion, and apoptosis (P < 0.05 or P < 0.01; Figure 6(b)–(f)). Taken together, our results demonstrated that H2O2 induced oxidative damage via upregulating BNIP3.

Figure 6.

BNIP3 modulated H2O2-induced oxidative damage: (a) BNIP3 mRNA and protein expression were analyzed by qRT-PCR or Western blot in EA.hy926 cells after transfection. (b) Cell viability was detected with CCK-8 assay. (c) Relative migration was assessed by a modified Boyden chamber. (d) A BD BioCoat Matrigel invasion was performed to analyze the invasive behavior. (e) Apoptotic cells were observed with flow cytometry after staining with Annexin V-FITC/PI. (f) Protein levels were examined with Western blot assay. EA.hy926 cells were treated with 150 μM H2O2 for 2 h and/or pre-incubated with/without 20 μM Rb1 for 1 h after transfection.

BNIP3: BCL2/adenovirus E1B 19-kDa interacting protein 3; sh: short hairpin; NC: negative control; qRT-PCR: quantitative reverse transcription polymerase chain reaction.

##P < 0.01 compared to pEX; ++P < 0.01 compared to sh-NC in (a). *P < 0.05, **P < 0.01, or ***P < 0.001 compared to control cells; #P < 0.05 or ##P < 0.01 compared to H2O2 + pEX cells; +P < 0.05 compared to H2O2 + sh-BNIP3 cells. n = 3–5.

H2O2 blocked mTOR and nuclear factor-κB signaling pathways via upregulating BNIP3

BNIP3 participates in H2O2-induced autophagic cell death by suppressing mTOR signaling pathway.[17] As for nuclear factor-κB (NF-κB) signaling pathway, a study revealed that NF-κB extends to the active transcriptional repression of the death factor BNIP3.[18] In this study, we found that the phosphorylation of S6 and mTOR was mitigated by H2O2 and BNIP3 overexpression further reduced S6 and mTOR phosphorylation. However, BNIP3 silence enhanced the phosphorylated expression of S6 and mTOR (Figure 7(a)). In addition, we also observed that the phosphorylation of IκBα and p65 was inhibited by H2O2 and upregulated BNIP3 further relieved the phosphorylation of IκBα and p65, while BNIP3 knockdown restored the phosphorylation of IκBα and p65 (Figure 7(b)). From the aforementioned results, we considered that H2O2 inactivated mTOR and NF-κB signaling pathways by upregulating BNIP3.

Figure 7.

Depletion of BNIP3 mediated S6, mTOR, IκBα, and p65 phosphorylation: (a) S6, p-S6, mTOR, p-mTOR, (b) IκBα, p-IκBα, p65, and p-p65 were quantified with Western blot assay. EA.hy926 cells were treated with 150 μM H2O2 for 2 h and/or pre-incubated with/without 20 μM Rb1 for 1 h after transfection. n = 3.

mTOR: mammalian targets of rapamycin; IκBα: inhibitor of nuclear factor κ-B α; BNIP3: BCL2/adenovirus E1B 19-kDa interacting protein 3; NC: negative control; sh: short hairpin; p: phospho.

Discussion

Multiple natural compounds, derived from plants, have been excavated for their application in alleviating oxidative injury. The anti-oxidative function of Rb1 has been elaborated in 6-hydroxydopamine-induced oxidative stress.[19] In this study, we found that Rb1 protected EA.hy926 cells against H2O2-induced oxidative injury by upregulating miR-210. Furthermore, we noticed that miR-210 negatively modulated BNIP3 and consequently mediated H2O2-induced oxidative injury via mTOR and NF-κB signaling pathways.

Previously published research has implied that H2O2 significantly decreased antioxidant enzyme activities.[20] Another important effect of H2O2 on oxidation is the generation of ROS, which has been validated to mediate apoptosis in an intrinsic manner.[21] Rb1, containing a tetracyclic triterpene skeleton with an aglycone, is a major component of ginseng. Its antioxidant activity has been proved in rat articular chondrocytes under H2O2-induced oxidative stress.[22] We found that Rb1 efficaciously restored cell viability, migration, and invasion, meanwhile repressed apoptosis in H2O2-treated endothelial cells. Furthermore, Rb1 repressed the activation of caspase-3 and caspase-9. It has been proved that oxidative stress induces apoptosis in a caspase-3 or caspase-9-dependent manner.[23,24] Collectively, Rb1 protected EA.hy926 against H2O2-induced apoptosis in a caspase-3 and caspase-9-dependent manner.

In addition, we found that Rb1-treated EA.hy926 cells showed upregulatory expression of Bcl-2, while downregulatory level of Bax after being treated with H2O2. Bcl-2 family member Bcl-2 protein is the essential modulator of apoptosis, and it has been considered as an effective molecular therapeutic target because of the key role in the pathogenesis of various diseases.[25] Bax is another member of Bcl-2 family, and its importance has been lighted in executing apoptosis process associated with mitochondrial membrane permeabilization.[26] As a consequence, the alteration of Bcl-2 and Bax indicated that the protective role of Rb1 was associated with its anti-apoptotic properties. In addition, its anti-apoptotic activity might be ascribed to its free radical scavenging capacity, which effectively preserves the mitochondrial membrane.[27] Specifically, a preceding study suggested that Rb1 augments the cellular antioxidant defenses through endoplasmic reticulum-dependent heme oxygenase-1 induction via several signaling pathways.[19]

Cytoprotection of miR-210 has been studied in several injury cell models. It has been validated that insulin protects H9c2 rat cardiomyoblast cells against H2O2-induced injury through upregulating the expression of miR-210.[28] The potential mechanism is associated with its regulatory function on mitochondrial free radical.[29] Furthermore, miR-210 overexpression reduced the apoptosis of mesenchymal stem cells under oxidative stress.[13] Similarly, we found that low level H2O2-induced miR-210 was not significant, while Rb1 obviously aggrandized miR-210 expression. Besides, miR-210 silence abrogated the protective effects of Rb1 in H2O2-treated cells. As a consequence, we considered that the anti-apoptotic activity of Rb1 was through regulation of miR-210. However, it is still incompletely understood how Rb1 modulated miR-210 expression.

BNIP3 has been identified as a redox sensor where oxidative stress triggers homodimerization and activation of BNIP3.[14] The primary function of miRs in protein output is based on the conserved match between miRs and the 3′-UTR of its target genes.[30] Besides, it has been found that BNIP3 is a target gene of miR-210 in neural progenitor cells.[15] In this study, we further consolidated that miR-210 negatively mediated BNIP3 expression by targeting its 3′-UTR. In addition, BNIP3 silence ameliorated H2O2-induced injury, whereas BNIP3 overexpression expedited H2O2-induced injury. Actually, an anterior study showed that BNIP3 interacts with dynamin Opa1 and consequently leads to mitochondrial fragmentation and apoptosis.[31] Consistently, our results suggested that BNIP3 silence blocked the activation of caspase-3 and caspase-9, indicating that BNIP3 knockdown impeded H2O2-induced apoptosis.

Rb1 augmented mTOR and NF-κB signaling pathways under inflammatory environment, which exhibited the anti-inflammatory activity.[32] On the basis of this study, we promoted and inhibited BNIP3 expression via transfection for addressing whether Rb1 triggered mTOR and NF-κB signaling pathways via BNIP3. Empirical evidence shows that the apoptosis is induced by mTOR inhibitor.[33,34] Moreover, NF-κB has been found to participate in the cell protective process of Rb1. Accordingly, BNIP3, as the mTOR and NF-κB signaling inhibitor, has been demonstrated to promote oxidative injury of endothelial cells in this study.

The vulnerability of cells to apoptosis is accompanied by the activation of mTOR and NF-κB cascades under multitude adverse stresses.[35-37] A previous study revealed a bidirectional association between mTOR and NF-κB signaling transduction cascades, which renders them indispensable for the regulation of BNIP3 expression.[38] As a result, there might be a feedback regulation between BNIP3 and mTOR and NF-κB pathways. Collectively, we concluded that Rb1 might protect endothelial cells against H2O2-induced oxidative injury by activating mTOR and NF-κB signaling pathways via downregulating BNIP3.

According to the aforementioned results, we concluded that the anti-apoptosis property of Rb1 was through inhibition of BNIP3 via upregulating miR-210. Furthermore, BNIP3 was testified to be involved in the H2O2-induced apoptosis via modulating mTOR and NF-κB signaling pathways.