MicroRNA-128b mediates lipopolysaccharide-induced apoptosis via reactive oxygen species in human pulmonary microvascular endothelial cells

Long, Guangwen; Yang, Xiulin; Ji, Chunling; Dong, Yukang

doi:10.1016/j.clinsp.2022.100020

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Original articles • Clinics 77 • 2022 • https://doi.org/10.1016/j.clinsp.2022.100020 copy

MicroRNA-128b mediates lipopolysaccharide-induced apoptosis via reactive oxygen species in human pulmonary microvascular endothelial cells

Authorship SCIMAGO INSTITUTIONS RANKINGS

ABSTRACT

Objectives:

This study aimed to explore the effects of miR-128b in the regulation of Lipopolysaccharide (LPS) induced apoptosis.

Methods:

Human Pulmonary Microvascular Endothelial Cells (HPMECs) were transfected with an miR-128b inhibitor and stimulated with LPS for 24 h. FCM was performed to detect apoptosis and Reactive Oxygen Species (ROS) production. In addition, miRNA and caspase-3 expression levels were determined using real-time quantitative polymerase chain reaction and western blotting.

Results:

LPS significantly induced apoptosis and ROS production and upregulated miR-128b and caspase-3 expressions in HPMECs. However, LPS-induced effects were suppressed when an miR-128b inhibitor was used. Preincu-bation with NAC decreased the LPS-induced apoptosis of HPMECs.

Conclusions:

These effects were mediated by miR-128b via the caspase-3 pathway.

Keywords:
miR-128b; PRKD1; Apoptosis; Reactive oxygen species; Human pulmonary microvascular endothelial cells

Authorship

Guangwen Long ^**Corresponding author. E-mail address: longguangwen2006@163.com (G. Long).

Emergency Department of Internal Medicine, Guizhou Provincial People's Hospital, Guiyang, ChinaGuizhou Provincial People's HospitalChinaGuiyang, ChinaEmergency Department of Internal Medicine, Guizhou Provincial People's Hospital, Guiyang, China

http://orcid.org/0000-0003-1248-2468

Xiulin Yang

http://orcid.org/0000-0002-9547-1731

Chunling Ji

http://orcid.org/0000-0002-8899-4841

Yukang Dong

http://orcid.org/0000-0001-8893-8324

*Corresponding author. E-mail address: longguangwen2006@163.com (G. Long).

CRediT authorship contribution statement

Guangwen Long: Visualization, Writing – review & editing. Xiulin Yang: Visualization, Formal analysis, Investigation, Writing – review & editing. Chunling Ji: Formal analysis, Investigation. Yukang Dong: Formal analysis, Investigation.

Conflicts of interest

The authors declare no conflicts of interest.

SCIMAGO INSTITUTIONS RANKINGS

Figures | Tables

Figures (7)
Tables (1)

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Figure 1
Detection of Human Pulmonary Microvascular Endothelial Cell (HPMEC) apoptosis via FCM analysis. HPMECs were treated with Lipopolysaccharide (LPS) (1 and 10 μg/mL) for 24 h. Apoptosis was detected via FCM analysis. Apoptotic cells were expressed as annexin V- and PI-positive cells. (A) Reactive Oxygen Species (ROS) production was measured using 2.7-Dichlorofluorescein Diacetate (DCFHDA) (10 µM) via FCM analysis (n = 4). (C, D) Expression levels of miRNA, PRKD1, and AK2 were detected using real-time quantitative Polymerase Chain Reaction (qRT-PCR) and western blotting. HPMECs were evaluated after treatment with LPS (1 μg/mL) or PBS for 24 h. Gene and protein expression levels were normalized to those of the control group (n = 4). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Thumbnail

Figure 2
(A, B) miRNA expression using real-time quantitative Polymerase Chain Reaction (qRT-PCR). Human Pulmonary Microvascular Endothelial Cells (HPMECs) were evaluated after 24 h of transfection with an miR-128b inhibitor (50 nM) or NC (10 nM) treated with Lipopolysaccharide (LPS) (1 μg/mL). The gene and protein expression levels were normalized to those of the control group or NC (n = 4). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Thumbnail

Figure 3
(A-) Detection of human pulmonary microvascular endothelial cell (HPMEC) apoptosis via FCM analysis. HPMECs were treated with Lipopolysaccharide (LPS) (1 μg/mL) for 24 h. Apoptosis was analyzed via FCM analysis. Apoptotic cells were defined as annexin V- and PI-positive cells. (C, D) Expression levels of PRKD1 determined using real-time quantitative Polymerase Chain Reaction (PCR). HPMECs were evaluated after 24 h of transfection with an miR-128b inhibitor (50 nM) or NC (10 nM) and after 24 h treatment stimulus with LPS (1 μg/mL) (n = 4). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Thumbnail

Figure 4
(C) Reactive Oxygen Species (ROS) production via FCM analysis using 2,7-Dichlorofluorescein Diacetate (DCFHDA). ROS production in Human Pulmonary Microvascular Endothelial Cells (HPMECs) was evaluated after 24h of transfection with an miR-128b inhibitor (50 nM), NC (10 nM), or CRT0066101 (5 μM, 10 μM) and after 24 h treatment with Lipopolysaccharide (LPS) (1 μg/mL) (n = 4). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Thumbnail

Figure 5
(A, B). Detection of human pulmonary microvascular endothelial cell (HPMEC) apoptosis via FCM analysis. Apoptosis was detected after 24h of transfection with an miR-128b inhibitor (50 nM) or NC (10 nM) and after 24 h treatment with Lipopolysaccharide (LPS) (1 μg/mL) or NAC (n = 4). Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

Thumbnail

Figure 6
(A, B) Representative western blots of cleaved caspase-3 in Human Pulmonary microvascular Endothelial Cells (HPMECs) transfected with an miR-128b inhibitor and NC. HPMECs were treated with LPS (1 μg/mL) or NAC (10 μM) (n = 3).

Thumbnail

Figure 7
miR-128b expression levels in the peripheral blood samples of patients with Acute Respiratory Distress Syndrome (ARDS) (n = 10) and healthy controls (n = 10) at different hours after the onset of ARDS. ****p < 0.0001.

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Table 1
Clinical characteristics of ARDS patients.

Figure 1 Detection of Human Pulmonary Microvascular Endothelial Cell (HPMEC) apoptosis via FCM analysis. HPMECs were treated with Lipopolysaccharide (LPS) (1 and 10 μg/mL) for 24 h. Apoptosis was detected via FCM analysis. Apoptotic cells were expressed as annexin V- and PI-positive cells. (A) Reactive Oxygen Species (ROS) production was measured using 2.7-Dichlorofluorescein Diacetate (DCFHDA) (10 µM) via FCM analysis (n = 4). (C, D) Expression levels of miRNA, PRKD1, and AK2 were detected using real-time quantitative Polymerase Chain Reaction (qRT-PCR) and western blotting. HPMECs were evaluated after treatment with LPS (1 μg/mL) or PBS for 24 h. Gene and protein expression levels were normalized to those of the control group (n = 4). Data are represented as mean ± SEM. p < 0.05, p < 0.01, p < 0.001.

Figure 2 (A, B) miRNA expression using real-time quantitative Polymerase Chain Reaction (qRT-PCR). Human Pulmonary Microvascular Endothelial Cells (HPMECs) were evaluated after 24 h of transfection with an miR-128b inhibitor (50 nM) or NC (10 nM) treated with Lipopolysaccharide (LPS) (1 μg/mL). The gene and protein expression levels were normalized to those of the control group or NC (n = 4). Data are represented as mean ± SEM. p < 0.05, p < 0.01, p < 0.001.

Figure 3 (A-) Detection of human pulmonary microvascular endothelial cell (HPMEC) apoptosis via FCM analysis. HPMECs were treated with Lipopolysaccharide (LPS) (1 μg/mL) for 24 h. Apoptosis was analyzed via FCM analysis. Apoptotic cells were defined as annexin V- and PI-positive cells. (C, D) Expression levels of PRKD1 determined using real-time quantitative Polymerase Chain Reaction (PCR). HPMECs were evaluated after 24 h of transfection with an miR-128b inhibitor (50 nM) or NC (10 nM) and after 24 h treatment stimulus with LPS (1 μg/mL) (n = 4). Data are represented as mean ± SEM. p < 0.05, p < 0.01, p < 0.001.

Figure 4 (C) Reactive Oxygen Species (ROS) production via FCM analysis using 2,7-Dichlorofluorescein Diacetate (DCFHDA). ROS production in Human Pulmonary Microvascular Endothelial Cells (HPMECs) was evaluated after 24h of transfection with an miR-128b inhibitor (50 nM), NC (10 nM), or CRT0066101 (5 μM, 10 μM) and after 24 h treatment with Lipopolysaccharide (LPS) (1 μg/mL) (n = 4). Data are represented as mean ± SEM. p < 0.05, p < 0.01, p < 0.001, ****p < 0.0001.

Figure 5 (A, B). Detection of human pulmonary microvascular endothelial cell (HPMEC) apoptosis via FCM analysis. Apoptosis was detected after 24h of transfection with an miR-128b inhibitor (50 nM) or NC (10 nM) and after 24 h treatment with Lipopolysaccharide (LPS) (1 μg/mL) or NAC (n = 4). Data are represented as mean ± SEM. p < 0.05, p < 0.01, p < 0.001.

Figure 6 (A, B) Representative western blots of cleaved caspase-3 in Human Pulmonary microvascular Endothelial Cells (HPMECs) transfected with an miR-128b inhibitor and NC. HPMECs were treated with LPS (1 μg/mL) or NAC (10 μM) (n = 3).

Figure 7 miR-128b expression levels in the peripheral blood samples of patients with Acute Respiratory Distress Syndrome (ARDS) (n = 10) and healthy controls (n = 10) at different hours after the onset of ARDS. ****p < 0.0001.

Table 1 Clinical characteristics of ARDS patients.

Patient, n°	Age/Sex	Lung injury score	PaO₂/FiO₂ (mm Hg)	Coexisting conditions
01	55/F	2.5	96.6	Lung cancer
02	65/M	2.7	89.7	Coronary heart disease
03	43/M	2.4	135.6	Trauma
04	58/F	2.1	121.8	Lung cancer
05	74/M	2.5	105.8	Hypertension
06	41/M	2.3	125.2	Trauma
07	55/F	2.6	132.1	Coronary heart disease
08	74/F	2.3	145.2	Lung cancer
09	69/M	2.4	100.7	Coronary heart disease
10	77/M	2.6	97.8	Chronic renal disease

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Faculdade de Medicina / USP Rua Dr Ovídio Pires de Campos, 225 - 6 and., 05403-010 São Paulo SP - Brazil, Tel.: (55 11) 2661-6235 - São Paulo - SP - Brazil
E-mail: clinics@hc.fm.usp.br

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MicroRNA-128b mediates lipopolysaccharide-induced apoptosis via reactive oxygen species in human pulmonary microvascular endothelial cells

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[1] *Corresponding author. E-mail address: longguangwen2006@163.com (G. Long).