Figures index : Puerarin Attenuates Transforming Growth Factor Beta-1 Induced Hypertrophic Responses and Smad Proetin Upregulation in Neonatal Rat Cardiomyocytes : Science and Education Publishing

Figures index

American Journal of Biomedical Research. 2020, 8(2), 40-46 doi:10.12691/ajbr-8-2-3

Figure 1. Dosage optimization of TGF-β₁ to induce hypertrophic responses in primary neonatal rat cardiomyocytes (NRCMs). NRCMs were seeded at a density of 10⁶cells/well in 24-well plates and cultured overnight. After a 24- h incubation in serum-free medium, NRCMs were stimulated with 0, 0.1, 1, 3, or 10 μg/L of TGF-β₁ for 24 h. (A–F) Cellular RNA was stained with RNA-sensitive fluorescence probe propidium iodide (PI) after DNase treatment. Representative images are shown in (A–E). Scale bar: 100 μm. The fluorescent signals were measured in five randomly selected fields in each well (F). (G) Protein synthesis was evaluated by measuring the incorporation of [³H]-leucine. The radioactivity of [³H]-leucine incorporated into the trichloroacetic acid precipitable material was determined by β-scintillation counting. The mRNA levels of atrial natriuretic factor (ANF) (H) and β-myosin heavy chain (β-MHC) (I) were determined using quantitative real-time PCR (qRT-PCR). GAPDH was used as an internal control. Data are expressed as mean ± standard deviation (SD). ^*P < 0.05 vs. control; ^#P < 0.05 vs. 3 μg/L TGF-β₁ (n=7).

Figure 2. Smad protein expression in TGF-β1-stimulated NRCMs. NRCMs were treated with TGF-β1 (3 μg/L) for 15 min, 30 min, 1 h, 2 h, or 24 h. (A) The protein expressions of Smad2, phospho-Smad2 (pSmad2; Ser465/467), and total Smad2/3 were determined using Western blot analysis. (B) Quantification of (A). β-actin was used as an internal control. Data are expressed as mean ± SD. *P < 0.05 vs. control (n=7)

Figure 3. NRCMs were transfected with different amounts of Smad2siRNA. Images were acquired at 24 h after transfection using a fluorescence microscope. Representative images are shown

Figure 4. Puerarin abrogated TGF-β1-induced hypertrophic response in NRCMs. (A) NRCMs were treated with different doses of puerarin (0.1, 1, or 5 g/L) for 24 h in the presence of TGF-β1 (3 μg/L). Protein synthesis in NRCMs was evaluated by measuring [³H]-leucine incorporation. (B) NRCMs were transfected with different amounts of Smad2siRNA (0.6, 1, or 1.4 μL). After 24 h of transfection, untransfected cells were treated with TGF-β1 (3 μg/L) individually or in combination with puerarin (1 g/L) for 24 h, and Smad2siRNA-transfected NRCMs were treated with TGF-β1 (3 μg/L) for 24 h. Protein synthesis was evaluated by measuring [³H]-leucine incorporation. (C) NRCMs were transfected with 1 μL of Smad2siRNA. After 24 h of transfection, untransfected cells were treated with TGF-β1 (3 μg/L) individually or in combination with puerarin (1 g/L) for 24 h, and Smad2siRNA -transfected cells were treated with TGF-β1 (3 μg/L) for 24 h. The mRNA levels of ANF and β-MHC were determined using qRT-PCR. GAPDH was used as an internal control. Data are expressed as mean ± SD. *P<0.05vs.control; #P<0.01vs.TGF-β₁ group; &P<0.05vs. puerarin group (n=6)

Figure 5. Puerarin attenuated TGF-β_1-induced Smad expression in NRCMs NRCMs were transfected with 1 μL of Smad2siRNA. After 24 h of transfection, untransfected cells were treated with TGF-β₁ (3 μg/L) individually or in combination with puerarin (1 g/L) for 24 h, and Smad2siRNA-transfected cells were treated with TGF-β₁(3 μg/L) for 24 h. Untreated cells were used as a control. mRNA (A) and protein (B) expression of Smad2, pSmad2, and total Smad2/3 were determined using qRT-PCR and Western blot analysis, respectively. (C) Quantification of (B). Data are expressed as mean ± SD. *P < 0.05 vs. control; #P < 0.01 vs. TGF-β1 group; &P < 0.05 vs. puerarin group (n=6)