Effect Of Oleanolic Acid On Neonatal Mice Cardiac Fibroblasts Proliferation Induced By Angiotensin Ⅱ And The Related Signaling Mechanism

Objective:To investigate the inhibitive effect of oleanolic acid(OA)on neonatal mice cardiac fibroblasts(cFs)proliferation induced by angiotension II(Ang II) and to explore the related mechanism.Methods:1. The culture and identification of cardiac fibroblasts: neonatal mice myofibroblasts were obtained from the heart of suckling mice through enzymatic digestion and differential adhesion method and identity of myofibroblasts were verified though aspecific antibodies immunofluorescence method; 2.To observe the effect of OA on c Fs: the experimental groups: ①control group; ②10μm OA group; ③20μm OA group; ④40μm OA group; ⑤60μm OA group; ⑥80μm OA group; ⑦100μm OA group; Proliferation of cFs was measured by MTT assay in 12 h, 24 h, 48 h, 72 h, 96h; 3. To observe the effect of OA on cFs: the experimental groups: ①control group; ②10-5mol/L AngII group;③10-6 mol/L AngII group; ④10-7 mol/L Ang II group; ⑤10-8 mol/L AngII group, Proliferation of cFs was measured by MTT assay in 24h、48h、72h、96h; 4. To observe the effect of OA on cardiac fibroblasts proliferation induced by angiotensin II: After cFs previously treated with AngII, spare, experimental groups as follows: ①control group; ②AngII(10-7mol / L) group; ③AngII(10-7mol / L) + OA(50μm) group; ④AngII(10-7mol / L) + OA(50μm) + ZnppIX(10μm, HO-1 activity in blocking agent); After incubation for 36 hours;Proliferation of cell was measured by MTT assay; The expression of Nrf2 、 HO-1mRNA in ①②③group was determined by fluorescence quantitative RT-PCR; 5. Screening effective lentiviruses: experimental groups as follows: ①control group, ②Nrf2 interference lentivirus group, ③empty virus group; After incubation for 96 hours, direct observation of GPF chromogenic by fluorescence coloration and detecting the expression of Nrf2 in cells by fluorescent quantitative RT-PCR method. 6.Nrf2 lentiviral intervention: The cFs induced by angiotension II after virus infection, spare, divided into: control group; ① ②AngII(10-7mol / L) group; ③AngII(10-7mol / L) + OA(50μm) group; ④OA(50μm) + AngII(10-7mol / L) + empty virus group; ⑤OA(50μm) + Ang II(10-7mol / L) + Nrf2 interference lentivirus group; After incubation for 36 hours; Proliferation of cell was measured by MTT assay; The expression of Nrf2 、 HO-1 mRNA in cell by fluorescence quantitative RT-PCR; Results:(1) 10-100μm concentration of OA had no significant effect on the activity of cFs;(2) 10-8, 10-7, 10-6, 10-5mol/L concentration of AngII can promote the proliferation of cFs, In the dependence of concentration and time, the strongest proliferation effect of AngII concentration in the 10-7mol/L and 24 h.(3) OA can significantly inhibit the proliferation cFs induced by AngII and promote the expression of Nrf2, HO-1mRNA, while effect of OA inhibition significantly reduced after adding ZnPPIX.(4) Nrf2 interference lentiviral infection cFs, visible green by fluorescence microscope; and the expression of Nrf2 m RNA decreased than the empty virus group in cells.(5) OA inhibited the proliferation of cFs induced by AngII significantly reduced after Nrf2 interference and the expression of HO-1 mRNA was significantly reduced. Conclusions:(1) OA can inhibited proliferation c Fs induced by AngII and promote the expression of Nrf2,HO-1 mRNA.(2) The nuclear transcription factor Nrf2 involved in regulating expression of HO-1 m RNA when OA effects AngII-induced cFs.(3) OA inhibits CFs proliferation induced by angiotension II though regulating the expression of HO-1.This study shows that OA inhibits neonatal mice cardiac fibroblasts proliferation induced by angiotension II, possibly through activation of nuclear transcription Nrf2 and subsequent upregulation of HO-1 expression.