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

Abstract

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