下调血管生成素样蛋白7表达对血管紧张素Ⅱ介导的血管平滑肌细胞炎症反应的影响

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

目的

探讨RNA干扰血管生成素样蛋白7 （Angptl7）基因对血管紧张素Ⅱ（AngⅡ）诱导的血管平滑肌细胞（VSMC）炎症因子的影响及其作用机制。

方法

体外培养人VSMC，分为常规F12K培养基培养（对照）和1 μg/mL AngII培养24 h。VSMC用AngⅡ（1 μg/mL）处理24 h后，采用siRNA-Angptl7和阴性对照siRNA-NC在Lipofectamine 2000介导下转染VSMC。RT-qPCR检测mRNA表达水平；Griess反应测定一氧化氮（NO）含量；蛋白免疫印记法检测相关蛋白的改变；酶联免疫吸附法检测VSMC中炎症因子肿瘤坏死因子α（TNF-α）、白细胞介素-1β （IL-1β）和IL-6水平。多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验，两组间比较采用独立样本t检验。

结果

与对照比较，1 μg/mL AngⅡ处理可促进VSMC中Angptl7 mRNA （0.97±0.06比3.05±0.21）和蛋白表达（1.01±0.12比1.61±0.14），亦可促进VSMC中IL-1β[（45.21±8.10）比（126.17±11.77） pg/mL]、IL-6[（50.50±7.51）比（108.50±9.51）pg/mL]和TNF-α的表达[（60.77±9.58）比（185.67±17.35）pg/mL]，差异有统计学意义（P均< 0.01）。与对照和转染siRNA-NC相比，转染siRNA-Angptl7下调Angptl7蛋白表达（0.99±0.12，0.98±0.12比0.44±0.14，P < 0.01）。与AngⅡ干预组相比，siRNA-Angptl7降低AngⅡ介导的VSMC炎症反应相关蛋白TNF-α、IL-6和IL-1β的表达，核因子κB （NF-κB）/诱导型一氧化氮合酶（iNOS）/环氧化酶2 （COX-2）信号通路相关蛋白NF-κB、iNOS和COX-2表达及NO含量亦降低，差异有统计学意义（P均< 0.01）。与siRNA-NC相比，siRNA-Angptl7组AngⅡ诱导的VSMC炎症反应相关蛋白TNF-α （0.99±0.13比0.51±0.12）、IL-6 （1.00±0.12比0.38±0.05）和IL-1β的表达（0.99±0.14比0.48±0.11），NF-κB （1.00±0.10比0.42±0.08）、iNOS （1.02±0.12比0.42±0.10）和COX-2表达（1.00±0.11比0.52±0.12）均降低，NO含量[（54.78±2.76）比（18.08±3.61）μmol/L]亦降低，差异有统计学意义（P均< 0.01）。

结论

AngⅡ可通过Angptl7促进VSMC炎症反应，下调Angptl7蛋白表达可以抑制VSMC的炎症反应，其作用机制可能与抑制NF-κB/iNOS-COX-2信号通路有关。

关键词: 血管生成素样蛋白, 血管紧张素Ⅱ, 血管平滑肌细胞, 炎症因子, 小干扰RNA

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

表1 引物序列信息

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

表1 引物序列信息

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

图1 AngⅡ对VSMC Angptl7的影响注：a图为AngⅡ对VSMC Angptl7 mRNA表达的影响；b图为AngⅡ对VSMC Angptl7蛋白表达的影响；c图为siRNA-Angptl7对VSMC Angptl7蛋白的影响；与对照比较，^aP < 0.01；与siRNA-NC组比较，^bP < 0.01；ns为差异无统计学意义

图2 AngⅡ对VSMC炎症因子IL-1β、IL-6和TNF-α表达的影响注：a、b、c图分别为IL-1β、IL-6和TNF-α表达；与对照比较，^aP < 0.01

图3 siAngptl7对VSMC炎症相关因子的影响注：a图为转染siAngptl7对VSMC炎症因子蛋白表达的影响；b图为转染siAngptl7对VSMC NF-κB/iNOS-COX-2信号通路相关蛋白表达的影响

表2 siRNA-Angptl7对VSMC炎症因子表达的影响（ ± s）

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

表2 siRNA-Angptl7对VSMC炎症因子表达的影响（ ± s）

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

图4 siRNA-Angptl7对炎症因子蛋白和NF-κB/iNOS-COX-2信号通路相关蛋白表达的影响注：a图为siRNA-Angptl7对炎症因子蛋白表达的影响；b图为siRNA-Angptl7对NF-κB/iNOS-COX-2信号通路相关蛋白表达的影响

表3 下调Angptl7对AngⅡ诱导的VSMC炎症反应的影响（ ± s）

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

表3 下调Angptl7对AngⅡ诱导的VSMC炎症反应的影响（ ± s）

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

图5 下调Angptl7对NO含量的影响注：与AngⅡ组和siRNA-NC+AngⅡ组相比较，^aP < 0.01，ns为差异无统计学意义

图6 下调Angptl7蛋白表达抑制AngⅡ介导的VSMC炎症反应机制注：AngⅡ为血管紧张素Ⅱ；Angptl7为血管生成素样蛋白7；siRNA-Angptl7为血管生成素样蛋白7干扰RNA；TNF-α为肿瘤坏死因子α；IL-1β为白介素1β；IL-6白介素6；Receptor为受体；NF-KB为核因子κB；iNOS诱导型一氧化氮合酶；COX-2环氧合酶-2；VSMC为血管平滑肌细胞；inflammatory为炎症

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

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