AngⅡ通过激活Notch1/Sox2通路对肝星状细胞LX2的活化和增殖的作用

doi:10.3877/cma.j.issn.2095-1221.2018.03.006

目的

探讨AngⅡ通过靶向调控Notch1/Sox2肝星状细胞LX2细胞的增殖。

方法

通过CCK8检测AngⅡ对肝星状细胞增殖能力的影响，通过羟脯氨酸酸法检测AngⅡ对肝星状细胞胶原合成能力的影响，并通过脂质体转染siRNA-Notch1构建Notch1低表达细胞模型，通过CCK8检测敲低Notch1对AngⅡ诱导的肝星状细胞LX2增殖的影响，通过Western Blot检测敲低Notch1对AngⅡ诱导的肝星状细胞LX2蛋白表达的影响。所有的检测结果都进行生物学重复。采用方差分析和t检验进行统计学分析。

结果

CCK8结果显示，AngⅡ（5、10、20、40、80 nmol/L）预处理A450值分别为0.67±0.06、0.88±0.07、0.98±0.07、1.08±0.07、1.23±0.07，较对照组0.57±0.05上升，差异具有统计学意义（F = 45.76，P < 0.01），羟脯氨酸检查结果显示，AngⅡ（10、20、40 nmol/L）预处理组羟脯氨酸浓度分别为（2.60±0.20）、（3.47±0.25）、（4.17±0.21）mg/L，羟脯氨酸浓度较对照组（1.90±0.10）mg/L上升，差异具有统计学意义（F = 75.18，P < 0.01）。Western Blot结果显示，AngⅡ10、20、40 nmol/L组Notch1蛋白表达水平分别为0.20±0.02、0.54±0.04、0.82±0.03，与正常对照组0.11±0.02发生升高，差异具有统计学意义（F = 400.50，P < 0.01）。Notch1干扰后，CCK8结果显示，siRNA-Notch1+AngⅡ组（10、20、40 nmol/L）A450值分别为0.53±0.06、0.83±0.03、1.03±0.03，与siRNA-NC+ AngⅡ对照组0.97±0.06，1.43±0.06，1.73±0.06比较发生降低（P < 0.01）。进一步Western Blot结果显示，Notch1敲低组（AngⅡ+ siRNA-Notch1）Notch1、HES1和Sox2蛋白表达水平分别为1.47±0.12、0.77±0.06和0.50±0.10，分别与AngⅡ对照组2.83±0.15、2.20±0.10和1.17±0.06比较，差异具有统计学意义（P < 0.01）。

结论

AngⅡ通过激活Notch1/Sox2信号促进肝星状细胞LX2增殖。

关键词: 肝星状细胞, 血管紧张素Ⅱ, 细胞增殖, Notch1

Objective

To investigated the effect of AngiotensinⅡ(AngⅡ) on the proliferation and apoptosis of hepatic stellate cells (HSCs) LX2 by targeting Notch1.

Methods

The effect of AngⅡ on the proliferation of HSCs was detected by CCK8. The effect of AngII on the collagen synthesis ability of HSCs was detected by hydroxyproline acid method. The Notch1 low expression cell model was constructed by transfecting siRNA-Notch1 with liposome. The effect of Notch1 on the proliferation of LX2 cells induced by AngII was detected by CCK8. The effect of knockdown of Notch1 on the expression of LX2 protein in HSCs induced by AngⅡ was detected by Western Blot. All test results were biologically replicated. Statistical analysis was performed using the analysis of variance and t-test.

Results

CCK8 results showed that the A450 of AngII pretreatment (5, 10, 20, 40, and 80 nmol/L) were (0.67±0.06), (0.88±0.07), (0.98±0.07), (1.08±0.07), (1.23±0.07), which was significantly higher than that in the control group (0.57±0.05) (F = 45.76, P < 0.01). The hydroxyproline test results showed that hydroxyproline concentrations in LX2 AngII pretreatment (10, 20, 40 nmol/L) were (2.60±0.20), (3.47±0.25), and (4.17±0.21) mg/L, and the concentration of hydroxyproline was significantly higher than that of the control group (1.90±0.10) mg/L (F = 75.18, P < 0.01). Western Blot results showed that the expression levels of Notch1 protein in the AngⅡ (10, 20, and 40 nmol/L) groups (0.20±0.02, 0.54±0.04, 0.82±0.03) were significantly higher than those in the normal control group (0.11±0.02). (F = 400.50, P < 0.01). After Notch1 interference, CCK8 results showed that the A450 values in the siRNA-Notch1+AngⅡ group (10, 20, 40 nmol/ L) were (0.53±0.06), (0.83±0.03), (1.03±0.03), which was significantly lower than that in the siRNA-NC+AngⅡ control group (0.97±0.06), (1.43±0.06), (1.73±0.06) (P < 0.01). Further Western Blot results showed that the Notch1, HES1 and Sox2 protein expression levels in the Notch1 knockdown group (AngⅡ+siRNA-Notch1) (1.47±0.12, 0.77±0.06, 0.50±0.10) were significantly decreased, compared with the AngII control group (2.83±0.15, 2.20±0.10, 1.17 ± 0.06) (P < 0.01).

Conclusion

AngⅡpromote the proliferation of HSCs by activating Notch1/Sox2.

Key words: Hepatic stellate cells, AngiotensinⅡ, Proliferation, Notch1

表1 不同浓度AngⅡ处理后CCK8检测LX2细胞A450值比较（±s）

分组	样本数	A450值
NC	3	0.57±0.05
5 nmol/L	3	0.72±0.03^*
10 nmol/L	3	0.88±0.07^**
20 nmol/L	3	0.98±0.07^**
40 nmol/L	3	1.08±0.07^**
80 nmol/L	3	1.23±0.07^**
F值	?	45.76
P值	?	< 0.01

表1 不同浓度AngⅡ处理后CCK8检测LX2细胞A450值比较（±s）

分组	样本数	A450值
NC	3	0.57±0.05
5 nmol/L	3	0.72±0.03^*
10 nmol/L	3	0.88±0.07^**
20 nmol/L	3	0.98±0.07^**
40 nmol/L	3	1.08±0.07^**
80 nmol/L	3	1.23±0.07^**
F值	?	45.76
P值	?	< 0.01

表2 不同浓度AngⅡ处理后消化法检测LX2细胞羟脯氨酸浓度（mg/L，±s）

分组	样本数	羟脯氨酸浓度
NC	3	1.90±0.10
10 nmol/L	3	2.60±0.20^**
20 nmol/L	3	3.47±0.25^**
40 nmol/L	3	4.17±0.21^**
F值	?	75.18
P值	?	< 0.01

表2 不同浓度AngⅡ处理后消化法检测LX2细胞羟脯氨酸浓度（mg/L，±s）

分组	样本数	羟脯氨酸浓度
NC	3	1.90±0.10
10 nmol/L	3	2.60±0.20^**
20 nmol/L	3	3.47±0.25^**
40 nmol/L	3	4.17±0.21^**
F值	?	75.18
P值	?	< 0.01

图1 AngⅡ对肝星状细胞胞Notch1蛋白表达的影响

表3 不同浓度AngⅡ诱导Notch1干扰前后的肝星状细胞增殖率A450的变化（nmol/L ，±s）

分组	样本数	不同浓度
分组	样本数	0	10	20	40
siRNA-NC组	3	0.47±0.06	0.97±0.06	1.43±0.06	1.73±0.06
siRNA-Notch1组	3	0.45±0.05	0.53±0.06	0.83±0.03	1.03±0.03
t值	?	?	9.19	16.09	20.09
P值	?	?	0.0008	< 0.01	< 0.01

表3 不同浓度AngⅡ诱导Notch1干扰前后的肝星状细胞增殖率A450的变化（nmol/L ，±s）

分组	样本数	不同浓度
分组	样本数	0	10	20	40
siRNA-NC组	3	0.47±0.06	0.97±0.06	1.43±0.06	1.73±0.06
siRNA-Notch1组	3	0.45±0.05	0.53±0.06	0.83±0.03	1.03±0.03
t值	?	?	9.19	16.09	20.09
P值	?	?	0.0008	< 0.01	< 0.01

表4 干扰Notch1AngⅡ诱导的肝星状细胞蛋白表达的变化（±s）

分组	样本数	Notch1相对表达量	HES1相对表达量	Sox2相对表达量
NC组	3	1.07±0.12	1.06±0.11	0.57±0.06
AngⅡ组	3	2.83±0.15^a	2.20±0.10^a	1.17±0.06^a
AngⅡ+siRNA-Notch1组	3	1.47±0.12^b	0.77±0.06^b	0.50±0.10^b
F值	?	154.5	192.9	72.8
P值	?	< 0.01	< 0.01	< 0.01

表4 干扰Notch1AngⅡ诱导的肝星状细胞蛋白表达的变化（±s）

分组	样本数	Notch1相对表达量	HES1相对表达量	Sox2相对表达量
NC组	3	1.07±0.12	1.06±0.11	0.57±0.06
AngⅡ组	3	2.83±0.15^a	2.20±0.10^a	1.17±0.06^a
AngⅡ+siRNA-Notch1组	3	1.47±0.12^b	0.77±0.06^b	0.50±0.10^b
F值	?	154.5	192.9	72.8
P值	?	< 0.01	< 0.01	< 0.01

图2 干扰Notch1和AngⅡ对人肝星状细胞蛋白表达的影响

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Effect of Ang Ⅱ on the activation and proliferation of hepatic stellate cell LX2 by activating the Notch1/Sox2 pathway

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Effect of Ang Ⅱ on the activation and proliferation of hepatic stellate cell LX2 by activating the Notch1/Sox2 pathway