Effect of down-regulation of angiopoietin-like protein 7 expression on AngⅡ-mediated inflammatory response of vascular smooth muscle cells

doi:10.3877/cma.j.issn.2095-1221.2022.02.005

Abstract

Abstract:

Objective

To investigate the effect of RNA interference of angiopoietin-like protein 7 (Angptl7) gene on angiotensinⅡ (AngⅡ) -induced inflammatory factors in vascular smooth muscle cells (VSMC) and its mechanism.

Methods

Human VSMC were cultured in conventional F12K medium in vitro (control group) and were treated with 1 μg/mL AngⅡ for 24 h respectively. After VSMCs were treated with AngⅡ (1 μg/mL) for 24 h, VSMCs were transfected with siRNA-Angptl7 and negative control siRNA-NC under the mediation of Lipofectamine 2000. The mRNA expression level was detected by RT-qPCR; nitric oxide (NO) content was determined by Griess reaction; changes of related proteins were detected by immunoblotting the effect of gene intervention on the inflammatory response of VSMC was observed. The levels of inflammatory factors TNF-α, IL-1β and IL-6 in VSMCs were detected by enzyme-linked immunosorbent assay. Data among multiple groups were compared with one-way ANOVA, LSD-t test was used for further comparison between two groups, and independent sample t-test was used for comparison between two groups.

Results

Compared with the control group, 1 μg/mL AngⅡtreatment could significantly increase the expression of Angptl7 mRNA (0.97±0.06 vs 3.05±0.21) and protein (1.01±0.12 vs 1.61±0.14) in VSMCs, and also promote the expression of IL-1β [ (45.21±8.10) vs (126.17±11.77) pg/mL], IL-6 [ (50.50±7.51) vs (108.50±9.51) pg/mL] and TNF-α [ (60.77±9.58) vs (185.67±17.35) pg/mL] in VSMCs, and the difference was statistically significant (P < 0.01) . Transfection of siRNA-Angptl7 down-regulated Angptl7 protein expression compared with control and transfected siRNA-NC groups (0.99±0.12, 0.98±0.12 vs 0.44±0.14, P < 0.01) . Compared with AngⅡ intervention group, siRNA-Angptl7 reduced the expression of AngⅡ-mediated VSMC inflammatory response-related proteins TNF-α, IL-6, IL-1β, Nuclear factor-κB (NF-κB) /inducible nitric oxide synthase (iNOS) /cyclooxygenase-2 (COX-2) signaling pathway-related protein NF-κB, iNOS and COX-2 expressions and NO content were also decreased, and the differences were statistically significant (all P < 0.01) . Compared with the siRNA-NC group, the AngⅡ-induced VSMCs inflammatory response-related proteins TNF-α (0.99±0.13 vs 0.51±0.12) , IL-6 (1.00±0.12 vs 0.38±0.05) , IL-1β (0.99±0.14 vs 0.48±0.11) in siRNA-Angptl7 group, NF-κB (1.00±0.10 vs 0.42±0.08) , iNOS (1.02±0.12 vs 0.42±0.10) , and COX-2 (1.00±0.11 vs 0.52±0.12) expressions were lower, and the NO content [ (54.78±2.76) vs (18.08±3.61) μmol/L] also decreased, and the differences were statistically significant (all P < 0.01) .

Conclusion

AngⅡ can promote the inflammatory response of VSMC through Angptl7. Downregulating the expression of Angptl7 gene can inhibit the inflammatory response of VSMCs, and its mechanism may be related to the inhibition of NF-κB/iNOS-COX-2 signaling pathway.

Key words: Angiopoietin-like protein, AngiotensinⅡ, Vascular smooth muscle cells, Inflammatory factors, RNA interference

Gonghao Li, Yanli Zhao, Zhongxing Peng, Delu Yin, Yunfeng Zhao. Effect of down-regulation of angiopoietin-like protein 7 expression on AngⅡ-mediated inflammatory response of vascular smooth muscle cells[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2022, 12(02): 93-99.

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References 37

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基因	引物序列（5'-3'）	预期扩增产物长度（bp）
Angptl7	上游TAGAGATGGAGGACTGGGAGG	198
	下游TGCACACTTGTCCAAGCAG
GAPDH	上游ACTTTGGCATTGTGGAAGG	152
	下游TGTGAGGGAGATGCTCAGTG

基因	引物序列（5'-3'）	预期扩增产物长度（bp）
Angptl7	上游TAGAGATGGAGGACTGGGAGG	198
	下游TGCACACTTGTCCAAGCAG
GAPDH	上游ACTTTGGCATTGTGGAAGG	152
	下游TGTGAGGGAGATGCTCAGTG

分组	实验次数	TNF-α	IL-1β	IL-6	NF-κB	COX-2	iNOS
siRNA-NC	3	1.00±0.09	1.01±0.09	1.00±0.09	0.99±0.08	1.00±0.10	1.00±0.10
siRNA-Angptl7	3	1.01±0.10	1.02±0.09	1.01±0.11	1.00±0.10	1.00±0.11	1.00±0.10
t值		-0.087	-0.103	-0.057	-0.129	-0.039	-0.012
P值		0.935	0.923	0.957	0.904	0.970	0.991

分组	实验次数	TNF-α	IL-1β	IL-6	NF-κB	COX-2	iNOS
siRNA-NC	3	1.00±0.09	1.01±0.09	1.00±0.09	0.99±0.08	1.00±0.10	1.00±0.10
siRNA-Angptl7	3	1.01±0.10	1.02±0.09	1.01±0.11	1.00±0.10	1.00±0.11	1.00±0.10
t值		-0.087	-0.103	-0.057	-0.129	-0.039	-0.012
P值		0.935	0.923	0.957	0.904	0.970	0.991

分组	实验次数	TNF-α	IL-1β	IL-6	NF-κB	COX-2	iNOS
AngⅡ	3	1.00±0.11	1.01±0.11	1.00±0.12	1.00±0.10	1.00±0.12	1.00±0.12
siRNA-NC+AngⅡ	3	0.99±0.13	1.00±0.12	0.99±0.14	1.00±0.10	1.00±0.11	1.02±0.12
siRNA-Angptl7+AngⅡ	3	0.51±0.12^ab	0.38±0.05^ab	0.48±0.11^ab	0.42±0.08^ab	0.52±0.12^abbc	0.42±0.10^ab
F值		16.238	17.497	41.261	39.701	17.419	25.804
P值		0.004	0.003	< 0.001	< 0.001	0.003	0.001

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