Expression and effect of Slit2/Robo4 signaling pathway in vitro model of transfusion related acute lung injury

doi:10.3877/cma.j.issn.1674-6880.2018.03.001

Abstract

Abstract:

Objective

To investigate the expression and effect of Slit2/Robo4 signaling pathway in vitro model of transfusion related acute lung injury (TRALI).

Methods

A two-hit model of polymorphonuclear neutrophils (PMNs)-mediated human pulmonary microvascular endothelial cells (HMVECs) damage was used as TRALI in vitro model. After the HMVECs were incubated for 0, 0.5, 1, 2, 4, and 6 h, the protein expressions of Robo4 and vascular endothelial cadherin (VE-cadherin) were detected by Western-blotting, and the mRNA expressions of Robo4 and Slit2 were analyzed by reverse transcription PCR (RT-PCR). The permeability of HMVECs was detected by in vitro experiment of endothelial cell permeability. Then the HMVECs were incubated with 0, 0.5, 1, 4, 10, and 20 μg/L Slit2-N for 24 h. The integrity and permeability of HMVECs were detected.

Results

In vitro model of TRALI, the mRNA expressions of Robo4 and Slit2 both showed significant differences at different time points (F=12.880, 11.060; both P < 0.001); they were much lower at 2 h (0.72 ± 0.04, 0.78 ± 0.05), 4 h (0.49 ± 0.04, 0.49 ± 0.06), and 6 h (0.34 ± 0.03, 0.43 ± 0.11) than those at 0 h (1.29 ± 0.06, 1.40 ± 0.09; all P < 0.05). The protein expression of Robo4 showed significant difference at each time point (F=11.560, P < 0.001); it decreased at 2, 4, 6 h (0.99 ± 0.04, 0.66 ± 0.03, 0.45 ± 0.04) in comparing with 0 h (1.44 ± 0.04, all P < 0.05). In addition, the protein expression of VE-cadherin at each time point was significantly different (F=9.667, P < 0.001); its expression decreased significantly at 2, 4, 6 h (0.91 ± 0.08, 0.78 ± 0.05, 0.50 ± 0.04) in comparing with 0 h (1.46 ± 0.09, all P < 0.05). The vitro experiment of endothelial cell permeability revealed that the permeability of HMVECs was significant different at each time point (F=21.940, P < 0.001); as comparing with 0 h (1.42 ± 0.16), the permeability of HMVECs was significantly higher at 2, 4, 6 h (4.00 ± 0.35, 5.70 ± 1.71, 10.02 ± 2.24; all P < 0.05). After adding the exogenous Slit2-N, the expression of VE-cadherin protein was statistically significantly different (F=13.220, P < 0.001); it increased in the 4, 10, 20 μg/L Slit2-N groups (1.19 ± 0.35, 1.49 ± 0.13, 2.12 ± 0.21) as comparing with the 0 μg/L Slit2-N group (0.41 ± 0.08, all P < 0.05). The vitro experiment of endothelial cell permeability revealed that the permeability of HMVECs was significant different (F=19.430, P < 0.001); the permeability of HMVECs was significantly lower in the 4, 10, 20 μg/L Slit2-N groups (4.2 ± 1.1, 2.1 ± 0.7, 1.8 ± 0.8) as comparing with the 0 μg/L Slit2-N group (10.0 ± 2.2, all P < 0.05).

Conclusion

The Slit2/Robo4 signaling pathway may be involved in the pathophysiological process of TRALI. Exogenous Slit2-N may modulate the integrity and permeability of HMVECs in vitro TRALI model, and become a promising candidate for developing novel therapies against TRALI.

Key words: Transfusion related acute lung injury, Slit2/Robo4, VE-cadherin, Human pulmonary microvascular endothelial cell

Dazhen Wei, Benji Wang, Mengxiang Lin, Xianyang Guo, Bihuan Cheng, Yuqiang Gong, Binyu Ying. Expression and effect of Slit2/Robo4 signaling pathway in vitro model of transfusion related acute lung injury[J]. Chinese Journal of Critical Care Medicine(Electronic Edition), 2018, 11(03): 145-150.

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References 20

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各时间点	细胞浓度（个/mL）	Robo4蛋白	Robo4 mRNA	Slit2 mRNA
0 h	1 × 10⁶	1.44 ± 0.04	1.29 ± 0.06	1.40 ± 0.09
0.5 h	1 × 10⁶	1.30 ± 0.04	1.26 ± 0.08	1.34 ± 0.05
1 h	1 × 10⁶	1.24 ± 0.07	0.94 ± 0.09	1.15 ± 0.19
2 h	1 × 10⁶	0.99 ± 0.04^a	0.72 ± 0.04^a	0.78 ± 0.05^a
4 h	1 × 10⁶	0.66 ± 0.03^a	0.49 ± 0.04^a	0.49 ± 0.06^a
6 h	1 × 10⁶	0.45 ± 0.04^a	0.34 ± 0.03^a	0.43 ± 0.11^a
F值	?	11.560	12.880	11.060
P值	?	<0.001	<0.001	<0.001

各时间点	细胞浓度（个/mL）	Robo4蛋白	Robo4 mRNA	Slit2 mRNA
0 h	1 × 10⁶	1.44 ± 0.04	1.29 ± 0.06	1.40 ± 0.09
0.5 h	1 × 10⁶	1.30 ± 0.04	1.26 ± 0.08	1.34 ± 0.05
1 h	1 × 10⁶	1.24 ± 0.07	0.94 ± 0.09	1.15 ± 0.19
2 h	1 × 10⁶	0.99 ± 0.04^a	0.72 ± 0.04^a	0.78 ± 0.05^a
4 h	1 × 10⁶	0.66 ± 0.03^a	0.49 ± 0.04^a	0.49 ± 0.06^a
6 h	1 × 10⁶	0.45 ± 0.04^a	0.34 ± 0.03^a	0.43 ± 0.11^a
F值	?	11.560	12.880	11.060
P值	?	<0.001	<0.001	<0.001

各时间点	细胞浓度（个/mL）	VE-cadherin蛋白	HMVECs通透性
0h	1 × 10⁶	1.46 ± 0.09	1.42 ± 0.16
0.5 h	1 × 10⁶	1.31 ± 0.08	1.61 ± 0.49
1h	1 × 10⁶	1.13 ± 0.05	2.06 ± 0.21
2h	1 × 10⁶	0.91 ± 0.08^a	4.00 ± 0.35^a
4h	1 × 10⁶	0.78 ± 0.05^a	5.70 ± 1.71^a
6h	1 × 10⁶	0.50 ± 0.04^a	10.02 ± 2.24^a
F值	?	9.667	21.940
P值	?	<0.001	<0.001

各时间点	细胞浓度（个/mL）	VE-cadherin蛋白	HMVECs通透性
0h	1 × 10⁶	1.46 ± 0.09	1.42 ± 0.16
0.5 h	1 × 10⁶	1.31 ± 0.08	1.61 ± 0.49
1h	1 × 10⁶	1.13 ± 0.05	2.06 ± 0.21
2h	1 × 10⁶	0.91 ± 0.08^a	4.00 ± 0.35^a
4h	1 × 10⁶	0.78 ± 0.05^a	5.70 ± 1.71^a
6h	1 × 10⁶	0.50 ± 0.04^a	10.02 ± 2.24^a
F值	?	9.667	21.940
P值	?	<0.001	<0.001

Slit2-N浓度（μg/L）	细胞浓度（个/mL）	VE-cadherin蛋白	HMVECs通透性
0	1 × 10⁶	0.41 ± 0.08	10.0 ± 2.2
0.5	1 × 10⁶	0.61 ± 0.06	10.1 ± 1.3
1	1 × 10⁶	0.68 ± 0.15	9.0 ± 1.4
4	1 × 10⁶	1.19 ± 0.35^a	4.2 ± 1.1^a
10	1 × 10⁶	1.49 ± 0.13^a	2.1 ± 0.7^a
20	1 × 10⁶	2.12 ± 0.21^a	1.8 ± 0.8^a
F值	?	13.220	19.430
P值	?	<0.001	<0.001

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