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15-HETE protects pulmonary artery smooth muscle cells against apoptosis via SIRT1 regulation during hypoxia
Biomedicine & Pharmacotherapy 108 (2018) 325–330
Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
15-HETE protects pulmonary artery smooth muscle cells against apoptosis via SIRT1 regulation during hypoxia Fujun Lia,b, Yanqiu Youc, Hui Zhua,
T
⁎
a
Deparment of Physiology of Harbin Medical University, No. 157 Baojian Road, Nangang District, Harbin, Heilongjiang, 150081, China Department of Anesthesiology, First Affiliated Hospital of Harbin Medical University, No. 199 Dazhi Street, Nangang District, Harbin, Heilongjiang, 150001, China c Department of Clinical Laboratory, Second Affiliated Hospital of Harbin Medical University, No. 246 Xuefu Road, Nangang District, Harbin, Heilongjiang, 150081, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: 15-HETE SIRT1 PASM Hypoxia Apoptosis
15-Hydroxyeicosatetraenoic acid (15-HETE) is produced by the catalytic metabolism of arachidonic acid by the enzyme 15-lipoxygenase. It is produced during hypoxia, and participates in the remodeling of pulmonary artery smooth muscle (PASM). Previous research has revealed that sirtuin 1 (SIRT1) involved in apoptosis in various cells and tissues. Herein, we attempted to determine whether 15-HETE counteracts SIRT1-promoted cell death in murine PASM cells (PASMCs). To verify this theory, we investigated changes in SIRT1 concentration in response to the counteraction of cell death by 15-HETE. We used western blotting and a terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, and investigated the survival, nuclear morphology, and mitochondrial potential of the cells. Our results revealed that 15-HETE promotes the transcription and translation of SIRT1. Moreover, 15-HETE increases viability and impaired mitochondrial depolarization, and promotes the expression of Bcl-2 and Bcl-xL in PASMCs without serum. The reactions mentioned above were eliminated by SIRT1 inhibitors (EX 527 and SIRT1 inhibitor IV). Our findings suggest that 15-HETE is crucial for the protection of PASMCs against cell death, and the SIRT1 pathway may provide a new strategy for pulmonary artery hypertension therapy.
1. Introduction Pulmonary artery hypertension (PAH) often leads to heart failure and mortality [1–3]. Pulmonary vascular remodeling (PVR) features medial hypertrophy and narrowing of the lumina of the small pulmonary arteries (PAs) arising from increased numbers of pulmonary artery smooth muscle cells (PASMCs) [4,5]. Malfunctioning cell death and cell proliferation result in an increase in the number of PASMCs (5), which contributes to PVR [6]. Chronic low levels of oxygen bring about PAH and suppress cell death in various cells such as PASMCs via unknown pathways [7,8]. Elucidation of the mechanisms by which these processes occur could provide innovative strategies for the treatment of PAH. Sirtuins (the name is derived from the homologous yeast gene “silent mating type information regulation 2″) are capable of deacetylating proteins at their lysine residues [9,10]. With regard to mammals, seven sirtuins (SIRT1–7) have been found in various subcellular compartments [11,12]. Sirtuins modulate the functions of various substrate proteins, thereby modulating transcription, metabolism, the stability of the genome, and viability [13,14]. Research has demonstrated the
⁎
expression of constitutive SIRT1 in the epithelia of large airways, and in smooth muscle cells (SMCs), macrophages, and the endothelial cells of vessels [15–17]. SIRT1 expression is stimulated in multiple cells in response to treatment with lipopolysaccharides, chronic hypoxia, proinflammatory cytokines, sepsis, or septic shock [18,19]. This study aimed to determine whether 15-hydroxyeicosatetraenoic acid (15-HETE) counteracts SIRT1-promoted cell death in murine PASM cells (PASMCs). 2. Materials and methods 2.1. Animals We used adult male Sprague-Dawley (SD) rats (4–6 weeks) (Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China). The animal study protocol was approved by the Committee on the Ethics of Animal Experiments of Deparment of Physiology of Harbin Medical University.
Corresponding author. E-mail addresses: [email protected] (F. Li), [email protected] (Y. You), [email protected] (H. Zhu).
https://doi.org/10.1016/j.biopha.2018.07.166 Received 6 June 2018; Received in revised form 27 July 2018; Accepted 31 July 2018 0753-3322/ © 2018 Published by Elsevier Masson SAS.
Biomedicine & Pharmacotherapy 108 (2018) 325–330
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2.2. Cell culture and reagents Primary cultivated PASMCs were generated from tissues obtained from rats (4–6 weeks). The dissociated cells were centrifuged, and then resuspended in DMEM with 20% fetal bovine serum (FBS) in a 37℃, 5% CO2 humidified incubator. 15-HETE was provided by Cayman Chemical (Ann Arbor, MI, USA), and was dissolved in ethanol before preservation in a nitrogen freezer. The SIRT1 inhibitors (EX 527 and SIRT1 inhibitor IV) were purchased from Santa Cruz Biotechnology.
Fig. 1. SIRT1 upregulation during hypoxia. PASMCs were subjected to hypoxia for various periods to investigate the changes in SIRT1 protein expression.
2.9. Statistical analysis
2.3. Western blotting (WB)
The results were presented as means ± SD. The data was analyzed by Student t-test or a one-way analysis of variance (ANOVA) by using Prism version 5. Values of p < 0.05 were considered statistically significant.
Protein concentrations were determined with the BCA Protein Quantitation Kit (Genscript, Piscataway, USA). Proteins were separated using 10% SDS-PAGE and blotted electrophoretically onto polyvinylidene difluoride membranes. Antibodies against SIRT1, Bcl-2, cleaved caspase-3, Bcl-xL, and β-actin (Santa Cruz Biotechnology, Dallas, USA) were used in this study. Horseradish peroxidase-linked secondary antibodies (Santa Cruz Biotechnology) were incubated with the membranes for 1 h and followed by chemiluminescent detection.
3. Results 3.1. SIRT1 upregulation during hypoxia WB was used to verify the influence of hypoxia on SIRT1 translation. We discovered that low levels of oxygen (2.5% O2/5% CO2/balance N2) triggered an obvious time-dependent upregulation of SIRT1 protein levels (Fig. 1).
2.4. Small interfering RNA (siRNA) design and transfection We cultivated the PASMCs until they reached 30–50% confluence. Incubation was carried out in DMEM without serum to simulate starvation. siRNA (200 pmole) was independently dissolved in Opti-MEM-1 medium without serum and mixed together. The admixture (siRNA/ transfection reagent) was incubated for 20 min at room temperature and was used to directly supplement the cells. The transfection reagent was removed 6–8 h after siRNA treatment. WB was carried out to evaluate the efficiency of protein silencing.
3.2. 15-HETE promoted the transcription and translation of SIRT1 in the cultured PASMCs WB and qRT-PCR were used to determine the influence of endogenous and exogenous 15-HETE on the expression of SIRT1 during normoxia and hypoxia. We found that hypoxia promoted the transcription and translation of SIRT1 compared with normoxia (Fig. 2A and B). The level of protein and mRNA were promoted if the cells received treatment with 15-HETE (Fig. 2A–D). However, the addition of nordihydroguaiaretic acid (NDGA), which is a selective arachidonate 15-lipoxygenase (15-LO) inhibitor, significantly eliminated the influence of 15-HETE (Fig. 2A–D). With regard to independent research, suppressing the production of 15-HETE using siRNA, or using NDGA to achieve 15-LO-2 knockdown, attenuates SIRT1 upregulation triggered by hypoxia, indicating that endogenous 15-HETE regulates SIRT1 expression triggered by hypoxia (Fig. 2E and F).
2.5. qRT-PCR RNA was isolated from the cultivated PASMCs. A complementary DNA (cDNA) synthesis kit was used for reverse transcription. The cDNA was amplified using a DNA thermal cycler (PerkinElmer, Bridgeville, PA, USA). β-Actin served as an internal reference during PCR quantification. 2.6. MTS assay
3.3. 15-HETE promoted PASMC survival via SIRT1 pathway
The PASMCs were placed in 96-well plates (1 × 104 cells/well). The wells were supplemented with a series of levels of idelalisib, and incubated for 72 h. A MTS assay kit was obtained from Promega (Fitchburg, WI, USA).
MTS assay was used to evaluate the influence of 5-HETE on PASMC survival. During normoxia and hypoxia, serum deprivation brought about a noticeable suppression of PASMC survival. However, 15-HETE defended survival following serum deprivation, which was markedly impaired by 2 μM EX 527 or 2 μM SIRT1 inhibitor IV during normoxia and hypoxia (Fig. 3A and B).
2.7. Apoptosis We cultivated the PASMCs in 6-well plates for 24 h using basal media. Subsequently, we added one of: SIRT1 inhibitor IV; EX 527; 15HETE; 15-HETE plus SIRT1 inhibitor IV; or 15-HETE plus EX 527. Apoptosis was determined by Hoechst 33,258 (Invitrogen) and Annexin V/propidium iodide (PI) staining.
3.4. 15-HETE stimulated the expression of Bcl-xL and Bcl-2 via SIRT1 pathway Bcl-xL and Bcl-2 prevent cell death. They were discovered on the membranes of mitochondria, and are related to mitochondrial activity, participating in cell death. Bcl-xL and Bcl-2 expression was promoted by 15-HETE. However, EX 527 and SIRT1 inhibitor IV partly impaired their influence (Fig. 4). Our research suggests that 15-HETE modulates mitochondrial membrane proteins that prevent cell death partially via an SIRT1 pathway, thereby preserving the mitochondria.
2.8. Evaluation of caspase-3 activity Caspase-3 activity was assessed by investigating the cleavage of the chromogenic caspase-3 substrate. We determined absorbance at 405 nm, and OD405 served as an indication for the amount of caspase-3. Proteins were subjected to WB. Incubation was carried out for 2 h at 37 °C. We used a spectrometer to determine the absorbance at 405 nm of the yellow pNA that had been cleaved from the precursor. The activity of caspase-3 was normalized to the total proteins of the cell lysates.
3.5. 15-HETE suppressed the stimulation of caspase-3 activation Caspase-3 is generated from a precursor named procaspase-3, which 326
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Fig. 2. 15-HETE increased SIRT1 protein and mRNA expression. SIRT1 protein (A) and mRNA (B) expression levels after treatment with either exogenous 15HETE (1 μM) or nordihydroguaiaretic acid (NDGA) during both normoxia and 48-h hypoxia. (C) SIRT1 protein and mRNA (D) expression levels after inhibiting endogenous 15-HETE with NDGA during 48-h hypoxia. (E) SIRT1 protein and mRNA (F) expression levels after blocking 15-HETE by knocking-down 15-LO-2 with siRNA during hypoxia. N = 6, *P < 0.05.
apoptosis, and was suppressed by 15-HETE in the PASMCs. Annexinpositive cells were quantified after 48 h of SD, as shown in Fig. 6B. 15HETE significantly reduced the number of annexin-positive cells triggered by SD. The protection provided by 15-HETE was impaired when SIRT1 was blocked with EX 527 or SIRT1 inhibitor IV.
undergoes cleavage in reaction to the death trigger via initiator caspases. The expression of procaspase-3 was used to indicate the activity of caspase-3. We found that 15-HETE suppressed caspase-3 activity and the expression of cleaved caspase-3 (Fig. 5A and B). 3.6. SIRT1 suppression eliminated the suppressive influence of 15-HETE on DNA fragmentation and nuclear shrinkage in the PASMCs
4. Discussion
We next investigated the influence of 15-HETE on the death of PASMCs using nuclear staining. Our research suggested that exogenous 15-HETE markedly reduced the number of cells with aberrant nuclei content (condensation, crenation, and fractionation) arising from serum deprivation (SD) (Fig. 6A). Nevertheless, after blocking SIRT 1 with EX 527 or SIRT1 inhibitor IV, 15-HETE was no longer able to inhibit the alteration of nuclei conformation under SD condition in PASMCs (Fig. 6A). The flow cytometry assay revealed that SIRT1 contributed to
Hypoxia-induced pulmonary vasoconstriction and pulmonary remodeling are important factors related to both primary and secondary PH [20,21]. Although there is insufficient understanding of its etiology, it has recently been proved that hypoxia promotes the production of 15HETE. 15-HETE makes an important contribution to the constriction and remodeling of vessels [22–24]. We discovered that 15-HETE triggers the transcription and translation of SIRT1 in PASMCs during both normoxia and hypoxia. Our research revealed that 15-HETE promotes the expression of SIRT1 327
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Fig. 3. 15-HETE promoted the survival of pulmonary artery smooth muscle cells (PASMCs) in an SIRT1-dependent manner. (A) PASMC viability during normoxia was determined using an MTS assay. (B) PASMC viability during hypoxia. N = 6, *P < 0.05.
during hypoxia, and that exogenous 15-HETE is also able to promote the expression of SIRT1 during normoxia. Furthermore, the results suggest that 15-HETE promotes viability through SIRT1 during normoxia and hypoxia. Consequently, we focused on normoxia to gain a better understanding of the relationship between 15-HETE, SIRT1, and cell death. Moreover, SIRT1 inhibitors reinforce cell death in PASMCs, suggesting that the SIRT1 axis is crucial for the prevention of cell death by 15-HETE in murine PASMCs. Our research revealed that exogenous 15HETE suppresses the activity of caspase-3, whereas EX 527 and SIRT1 inhibitor IV prohibit the influence of 15-HETE on caspase-3 with regard to PASMCs. It is thought that exogenous 15-HETE suppresses cell death via an SIRT1-caspase-3 axis. Previous research has revealed that SD stimulates certain death reactions that cause the malfunction of mitochondria [25]. 15-HETE promotes the expression of Bcl-xL, which is found in the mitochondrial outer membrane, and regulates its stabilization. Mitochondrial signals are crucial to the inhibition of cell death via 15-HETE through an SIRT1 pathway in PASMCs. In our present study, we discovered noticeable mitochondrial depolarization subsequent to HETE exposure for 15 m,
Fig. 4. 15-HETE regulates Bcl-2 and Bcl-xL expression induced by serum deprivation via the SIRT1 pathway in PASMCs. The expression of Bcl-2 and Bcl-xL in rat PASMCs during normoxia.
Fig. 5. Exogenous 15-HETE regulated caspase-3 activation through SIRT1. (A) Caspase-3 activity was determined by cleavage of the Ac-DEVD-pNA substrate to pNA. (B) Cleaved caspase-3 expression in rat PASMCs after applying 15-HETE and SIRT1 inhibitors. N = 6, *P < 0.05. 328
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Fig. 6. Exogenous 15-HETE regulated apoptosis through SIRT1. (A) Apoptotic cells were investigated by nuclear staining. (B) Apoptosis was detected by flow cytometry in rat PASMCs after applying exogenous 15-HETE and SIRT1 inhibitors. N = 6, *P < 0.05.
Acknowledgment
which was impaired by SIRT1 inhibitors. A TUNEL assay revealed that 15-HETE reduced the number of apoptotic cells. The results indicate that the suppressing effect of 15-HETE on cell death is impaired by SIRT1 pathway inhibitors.
This work was supported by Scientific research project of the health planning committee of Heilongjiang (2016-030). References
5. Conclusion [1] E.M.T. Lau, E. Giannoulatou, D.S. Celermajer, M. Humbert, Epidemiology and treatment of pulmonary arterial hypertension, Nat. Rev. Cardiol. 14 (2017) 603–614. [2] S. Sahni, M. Ojrzanowski, S. Majewski, A. Talwar, Pulmonary arterial hypertension: a current review of pharmacological management, Pneumonol. Alergol. Pol. 84 (2016) 47–61. [3] H. Tsai, Y.K. Sung, V. de Jesus Perez, Recent advances in the management of pulmonary arterial hypertension, F1000Res 5 (2016) 2755. [4] J.A. Leopold, B.A. Maron, Molecular mechanisms of pulmonary vascular remodeling in pulmonary arterial hypertension, Int. J. Mol. Sci. 17 (2016) 761. [5] N.W. Morrell, S. Adnot, S.L. Archer, J. Dupuis, P.L. Jones, M.R. MacLean, I.F. McMurtry, K.R. Stenmark, P.A. Thistlethwaite, N. Weissmann, J.X. Yuan, E.K. Weir, Cellular and molecular basis of pulmonary arterial hypertension, J. Am. Coll. Cardiol. 54 (2009) S20–S31. [6] S. Malenfant, A.S. Neyron, R. Paulin, F. Potus, J. Meloche, S. Provencher, S. Bonnet, Signal transduction in the development of pulmonary arterial hypertension, Pulm.
Our findings suggest that 15-HETE promotes the transcription and translation of SIRT1, and that endogenous 15-HETE suppresses the death of PASMCs via an SIRT1 pathway. Although our research demonstrated that an SIRT1 pathway participates in the suppressive influence of 15-HETE on the death of PASMCs, more research is required to determine whether SIRT1 is stimulated by heme oxygenase-1 (HO-1) or other agents during hypoxia.
Conflicts of interest None. 329
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Contents lists available at ScienceDirect
Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
15-HETE protects pulmonary artery smooth muscle cells against apoptosis via SIRT1 regulation during hypoxia Fujun Lia,b, Yanqiu Youc, Hui Zhua,
T
⁎
a
Deparment of Physiology of Harbin Medical University, No. 157 Baojian Road, Nangang District, Harbin, Heilongjiang, 150081, China Department of Anesthesiology, First Affiliated Hospital of Harbin Medical University, No. 199 Dazhi Street, Nangang District, Harbin, Heilongjiang, 150001, China c Department of Clinical Laboratory, Second Affiliated Hospital of Harbin Medical University, No. 246 Xuefu Road, Nangang District, Harbin, Heilongjiang, 150081, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: 15-HETE SIRT1 PASM Hypoxia Apoptosis
15-Hydroxyeicosatetraenoic acid (15-HETE) is produced by the catalytic metabolism of arachidonic acid by the enzyme 15-lipoxygenase. It is produced during hypoxia, and participates in the remodeling of pulmonary artery smooth muscle (PASM). Previous research has revealed that sirtuin 1 (SIRT1) involved in apoptosis in various cells and tissues. Herein, we attempted to determine whether 15-HETE counteracts SIRT1-promoted cell death in murine PASM cells (PASMCs). To verify this theory, we investigated changes in SIRT1 concentration in response to the counteraction of cell death by 15-HETE. We used western blotting and a terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) assay, and investigated the survival, nuclear morphology, and mitochondrial potential of the cells. Our results revealed that 15-HETE promotes the transcription and translation of SIRT1. Moreover, 15-HETE increases viability and impaired mitochondrial depolarization, and promotes the expression of Bcl-2 and Bcl-xL in PASMCs without serum. The reactions mentioned above were eliminated by SIRT1 inhibitors (EX 527 and SIRT1 inhibitor IV). Our findings suggest that 15-HETE is crucial for the protection of PASMCs against cell death, and the SIRT1 pathway may provide a new strategy for pulmonary artery hypertension therapy.
1. Introduction Pulmonary artery hypertension (PAH) often leads to heart failure and mortality [1–3]. Pulmonary vascular remodeling (PVR) features medial hypertrophy and narrowing of the lumina of the small pulmonary arteries (PAs) arising from increased numbers of pulmonary artery smooth muscle cells (PASMCs) [4,5]. Malfunctioning cell death and cell proliferation result in an increase in the number of PASMCs (5), which contributes to PVR [6]. Chronic low levels of oxygen bring about PAH and suppress cell death in various cells such as PASMCs via unknown pathways [7,8]. Elucidation of the mechanisms by which these processes occur could provide innovative strategies for the treatment of PAH. Sirtuins (the name is derived from the homologous yeast gene “silent mating type information regulation 2″) are capable of deacetylating proteins at their lysine residues [9,10]. With regard to mammals, seven sirtuins (SIRT1–7) have been found in various subcellular compartments [11,12]. Sirtuins modulate the functions of various substrate proteins, thereby modulating transcription, metabolism, the stability of the genome, and viability [13,14]. Research has demonstrated the
⁎
expression of constitutive SIRT1 in the epithelia of large airways, and in smooth muscle cells (SMCs), macrophages, and the endothelial cells of vessels [15–17]. SIRT1 expression is stimulated in multiple cells in response to treatment with lipopolysaccharides, chronic hypoxia, proinflammatory cytokines, sepsis, or septic shock [18,19]. This study aimed to determine whether 15-hydroxyeicosatetraenoic acid (15-HETE) counteracts SIRT1-promoted cell death in murine PASM cells (PASMCs). 2. Materials and methods 2.1. Animals We used adult male Sprague-Dawley (SD) rats (4–6 weeks) (Shanghai SLAC Laboratory Animal Co., Ltd., Shanghai, China). The animal study protocol was approved by the Committee on the Ethics of Animal Experiments of Deparment of Physiology of Harbin Medical University.
Corresponding author. E-mail addresses: [email protected] (F. Li), [email protected] (Y. You), [email protected] (H. Zhu).
https://doi.org/10.1016/j.biopha.2018.07.166 Received 6 June 2018; Received in revised form 27 July 2018; Accepted 31 July 2018 0753-3322/ © 2018 Published by Elsevier Masson SAS.
Biomedicine & Pharmacotherapy 108 (2018) 325–330
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2.2. Cell culture and reagents Primary cultivated PASMCs were generated from tissues obtained from rats (4–6 weeks). The dissociated cells were centrifuged, and then resuspended in DMEM with 20% fetal bovine serum (FBS) in a 37℃, 5% CO2 humidified incubator. 15-HETE was provided by Cayman Chemical (Ann Arbor, MI, USA), and was dissolved in ethanol before preservation in a nitrogen freezer. The SIRT1 inhibitors (EX 527 and SIRT1 inhibitor IV) were purchased from Santa Cruz Biotechnology.
Fig. 1. SIRT1 upregulation during hypoxia. PASMCs were subjected to hypoxia for various periods to investigate the changes in SIRT1 protein expression.
2.9. Statistical analysis
2.3. Western blotting (WB)
The results were presented as means ± SD. The data was analyzed by Student t-test or a one-way analysis of variance (ANOVA) by using Prism version 5. Values of p < 0.05 were considered statistically significant.
Protein concentrations were determined with the BCA Protein Quantitation Kit (Genscript, Piscataway, USA). Proteins were separated using 10% SDS-PAGE and blotted electrophoretically onto polyvinylidene difluoride membranes. Antibodies against SIRT1, Bcl-2, cleaved caspase-3, Bcl-xL, and β-actin (Santa Cruz Biotechnology, Dallas, USA) were used in this study. Horseradish peroxidase-linked secondary antibodies (Santa Cruz Biotechnology) were incubated with the membranes for 1 h and followed by chemiluminescent detection.
3. Results 3.1. SIRT1 upregulation during hypoxia WB was used to verify the influence of hypoxia on SIRT1 translation. We discovered that low levels of oxygen (2.5% O2/5% CO2/balance N2) triggered an obvious time-dependent upregulation of SIRT1 protein levels (Fig. 1).
2.4. Small interfering RNA (siRNA) design and transfection We cultivated the PASMCs until they reached 30–50% confluence. Incubation was carried out in DMEM without serum to simulate starvation. siRNA (200 pmole) was independently dissolved in Opti-MEM-1 medium without serum and mixed together. The admixture (siRNA/ transfection reagent) was incubated for 20 min at room temperature and was used to directly supplement the cells. The transfection reagent was removed 6–8 h after siRNA treatment. WB was carried out to evaluate the efficiency of protein silencing.
3.2. 15-HETE promoted the transcription and translation of SIRT1 in the cultured PASMCs WB and qRT-PCR were used to determine the influence of endogenous and exogenous 15-HETE on the expression of SIRT1 during normoxia and hypoxia. We found that hypoxia promoted the transcription and translation of SIRT1 compared with normoxia (Fig. 2A and B). The level of protein and mRNA were promoted if the cells received treatment with 15-HETE (Fig. 2A–D). However, the addition of nordihydroguaiaretic acid (NDGA), which is a selective arachidonate 15-lipoxygenase (15-LO) inhibitor, significantly eliminated the influence of 15-HETE (Fig. 2A–D). With regard to independent research, suppressing the production of 15-HETE using siRNA, or using NDGA to achieve 15-LO-2 knockdown, attenuates SIRT1 upregulation triggered by hypoxia, indicating that endogenous 15-HETE regulates SIRT1 expression triggered by hypoxia (Fig. 2E and F).
2.5. qRT-PCR RNA was isolated from the cultivated PASMCs. A complementary DNA (cDNA) synthesis kit was used for reverse transcription. The cDNA was amplified using a DNA thermal cycler (PerkinElmer, Bridgeville, PA, USA). β-Actin served as an internal reference during PCR quantification. 2.6. MTS assay
3.3. 15-HETE promoted PASMC survival via SIRT1 pathway
The PASMCs were placed in 96-well plates (1 × 104 cells/well). The wells were supplemented with a series of levels of idelalisib, and incubated for 72 h. A MTS assay kit was obtained from Promega (Fitchburg, WI, USA).
MTS assay was used to evaluate the influence of 5-HETE on PASMC survival. During normoxia and hypoxia, serum deprivation brought about a noticeable suppression of PASMC survival. However, 15-HETE defended survival following serum deprivation, which was markedly impaired by 2 μM EX 527 or 2 μM SIRT1 inhibitor IV during normoxia and hypoxia (Fig. 3A and B).
2.7. Apoptosis We cultivated the PASMCs in 6-well plates for 24 h using basal media. Subsequently, we added one of: SIRT1 inhibitor IV; EX 527; 15HETE; 15-HETE plus SIRT1 inhibitor IV; or 15-HETE plus EX 527. Apoptosis was determined by Hoechst 33,258 (Invitrogen) and Annexin V/propidium iodide (PI) staining.
3.4. 15-HETE stimulated the expression of Bcl-xL and Bcl-2 via SIRT1 pathway Bcl-xL and Bcl-2 prevent cell death. They were discovered on the membranes of mitochondria, and are related to mitochondrial activity, participating in cell death. Bcl-xL and Bcl-2 expression was promoted by 15-HETE. However, EX 527 and SIRT1 inhibitor IV partly impaired their influence (Fig. 4). Our research suggests that 15-HETE modulates mitochondrial membrane proteins that prevent cell death partially via an SIRT1 pathway, thereby preserving the mitochondria.
2.8. Evaluation of caspase-3 activity Caspase-3 activity was assessed by investigating the cleavage of the chromogenic caspase-3 substrate. We determined absorbance at 405 nm, and OD405 served as an indication for the amount of caspase-3. Proteins were subjected to WB. Incubation was carried out for 2 h at 37 °C. We used a spectrometer to determine the absorbance at 405 nm of the yellow pNA that had been cleaved from the precursor. The activity of caspase-3 was normalized to the total proteins of the cell lysates.
3.5. 15-HETE suppressed the stimulation of caspase-3 activation Caspase-3 is generated from a precursor named procaspase-3, which 326
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Fig. 2. 15-HETE increased SIRT1 protein and mRNA expression. SIRT1 protein (A) and mRNA (B) expression levels after treatment with either exogenous 15HETE (1 μM) or nordihydroguaiaretic acid (NDGA) during both normoxia and 48-h hypoxia. (C) SIRT1 protein and mRNA (D) expression levels after inhibiting endogenous 15-HETE with NDGA during 48-h hypoxia. (E) SIRT1 protein and mRNA (F) expression levels after blocking 15-HETE by knocking-down 15-LO-2 with siRNA during hypoxia. N = 6, *P < 0.05.
apoptosis, and was suppressed by 15-HETE in the PASMCs. Annexinpositive cells were quantified after 48 h of SD, as shown in Fig. 6B. 15HETE significantly reduced the number of annexin-positive cells triggered by SD. The protection provided by 15-HETE was impaired when SIRT1 was blocked with EX 527 or SIRT1 inhibitor IV.
undergoes cleavage in reaction to the death trigger via initiator caspases. The expression of procaspase-3 was used to indicate the activity of caspase-3. We found that 15-HETE suppressed caspase-3 activity and the expression of cleaved caspase-3 (Fig. 5A and B). 3.6. SIRT1 suppression eliminated the suppressive influence of 15-HETE on DNA fragmentation and nuclear shrinkage in the PASMCs
4. Discussion
We next investigated the influence of 15-HETE on the death of PASMCs using nuclear staining. Our research suggested that exogenous 15-HETE markedly reduced the number of cells with aberrant nuclei content (condensation, crenation, and fractionation) arising from serum deprivation (SD) (Fig. 6A). Nevertheless, after blocking SIRT 1 with EX 527 or SIRT1 inhibitor IV, 15-HETE was no longer able to inhibit the alteration of nuclei conformation under SD condition in PASMCs (Fig. 6A). The flow cytometry assay revealed that SIRT1 contributed to
Hypoxia-induced pulmonary vasoconstriction and pulmonary remodeling are important factors related to both primary and secondary PH [20,21]. Although there is insufficient understanding of its etiology, it has recently been proved that hypoxia promotes the production of 15HETE. 15-HETE makes an important contribution to the constriction and remodeling of vessels [22–24]. We discovered that 15-HETE triggers the transcription and translation of SIRT1 in PASMCs during both normoxia and hypoxia. Our research revealed that 15-HETE promotes the expression of SIRT1 327
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Fig. 3. 15-HETE promoted the survival of pulmonary artery smooth muscle cells (PASMCs) in an SIRT1-dependent manner. (A) PASMC viability during normoxia was determined using an MTS assay. (B) PASMC viability during hypoxia. N = 6, *P < 0.05.
during hypoxia, and that exogenous 15-HETE is also able to promote the expression of SIRT1 during normoxia. Furthermore, the results suggest that 15-HETE promotes viability through SIRT1 during normoxia and hypoxia. Consequently, we focused on normoxia to gain a better understanding of the relationship between 15-HETE, SIRT1, and cell death. Moreover, SIRT1 inhibitors reinforce cell death in PASMCs, suggesting that the SIRT1 axis is crucial for the prevention of cell death by 15-HETE in murine PASMCs. Our research revealed that exogenous 15HETE suppresses the activity of caspase-3, whereas EX 527 and SIRT1 inhibitor IV prohibit the influence of 15-HETE on caspase-3 with regard to PASMCs. It is thought that exogenous 15-HETE suppresses cell death via an SIRT1-caspase-3 axis. Previous research has revealed that SD stimulates certain death reactions that cause the malfunction of mitochondria [25]. 15-HETE promotes the expression of Bcl-xL, which is found in the mitochondrial outer membrane, and regulates its stabilization. Mitochondrial signals are crucial to the inhibition of cell death via 15-HETE through an SIRT1 pathway in PASMCs. In our present study, we discovered noticeable mitochondrial depolarization subsequent to HETE exposure for 15 m,
Fig. 4. 15-HETE regulates Bcl-2 and Bcl-xL expression induced by serum deprivation via the SIRT1 pathway in PASMCs. The expression of Bcl-2 and Bcl-xL in rat PASMCs during normoxia.
Fig. 5. Exogenous 15-HETE regulated caspase-3 activation through SIRT1. (A) Caspase-3 activity was determined by cleavage of the Ac-DEVD-pNA substrate to pNA. (B) Cleaved caspase-3 expression in rat PASMCs after applying 15-HETE and SIRT1 inhibitors. N = 6, *P < 0.05. 328
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Fig. 6. Exogenous 15-HETE regulated apoptosis through SIRT1. (A) Apoptotic cells were investigated by nuclear staining. (B) Apoptosis was detected by flow cytometry in rat PASMCs after applying exogenous 15-HETE and SIRT1 inhibitors. N = 6, *P < 0.05.
Acknowledgment
which was impaired by SIRT1 inhibitors. A TUNEL assay revealed that 15-HETE reduced the number of apoptotic cells. The results indicate that the suppressing effect of 15-HETE on cell death is impaired by SIRT1 pathway inhibitors.
This work was supported by Scientific research project of the health planning committee of Heilongjiang (2016-030). References
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Our findings suggest that 15-HETE promotes the transcription and translation of SIRT1, and that endogenous 15-HETE suppresses the death of PASMCs via an SIRT1 pathway. Although our research demonstrated that an SIRT1 pathway participates in the suppressive influence of 15-HETE on the death of PASMCs, more research is required to determine whether SIRT1 is stimulated by heme oxygenase-1 (HO-1) or other agents during hypoxia.
Conflicts of interest None. 329
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