Fig 2: GA attenuates Nox2 expression in SHRs and angiotensin II-treated H9c2 cells. (A) The mRNA levels of Nox1, Nox2, and Nox4 were analyzed using qRT-PCR. The transcript levels have been normalized to 18 S and presented as a relative value. ***P < 0.001 versus WKY. # P < 0.05 and ### P < 0.001 versus SHR. (B) Representative immunoblots. Nox1, Nox2, and Nox4 protein expression in rat hearts from the WKY, SHR, and SHR+GA groups, was determined using western blot analysis. GAPDH was used as the loading control. (C) The protein expression of Nox1, Nox2, and Nox4 was quantified by densitometry. ***P < 0.001 versus WKY. # P < 0.05 versus SHR. (D) The mRNA levels of Nox1, Nox2, and Nox4 were determined using H9c2 cells treated with the vehicle (control), with vehicle + Ang II (1 µM), or with Ang II (1 µM) + GA (100 µM) for 9 h. The values are the mean ± SE of at least three independent experiments. **P < 0.01 versus vehicle-treated cells. # P < 0.05 versus angiotensin II-treated cells. NS, not significant.

Fig 3: Myocardial histopathology and protein expression of NOX1, NOX2 and NOX4. (A) Masson staining and CVF (magnification, ×200). (B) Sirius red staining and CVF (magnification, ×200). Collagen stained with Masson staining (blue) and Sirius red staining (red) are significantly increased in the DOX group compared with those in the CON group, which is attenuated in the DOX+Val group. (C) Myocardial protein expression of NOX1, NOX2 and NOX4 detected by western blotting. *P<0.05 vs. CON; †P<0.05 vs. DOX.CON, control; DOX, doxorubicin; Val, valsartan; NOX, NAD(P)H oxidase; CVF, collagen volume fraction.

Fig 4: Apoptosis in vitro in H9C2 cells post-treatment. (A) Apoptosis in H9C2 cells detected by flow cytometry. (B) Cardiomyocyte apoptosis was significantly increased in DOX-treated H9C2 cells compared with the control group, which was significantly reduced by DOX+Val and NOX2-siRNA and NOX4-siRNA, whereas apoptosis in DOX-treated H9C2 cells was further increased following NOX2 and NOX4 overexpression. *P<0.05 vs. control; †P<0.05 vs. DOX. DOX, doxorubicin; Val, valsartan; NOX, NAD(P)H oxidase; siRNA, small interfering RNA; OE, overexpressed.

Fig 5: The anti-fibrogenic activity of OVA in TGF-ß1-induced fibrotic LL29 cells. (A) Myofibroblast-associated and ECM proteins. For myofibroblast-associated and ECM proteins, LL29 cells were treated with 5 ng/mL TGF-ß1 with or without OVA for 48 h. The COL1A1, ?-SMA, fibronectin, and vimentin levels were determined. (B–C) TGF-ß/TßRs signaling-related proteins. For TGF-ß/TßRs signaling-related proteins, LL29 cells were treated with 5 ng/mL TGF-ß1 with or without OVA for 24 h. (B) The pTßRII, TßRII pTßRI, TßRI, pSMAD2, and SMAD2 levels were determined. (C) The ZEB1 and NOX4 levels were determined. Protein expression was characterized by western blot analysis. Fold changes relative to the vehicle control group are shown.