Rutin Protects against Pirarubicin‐Induced Cardiotoxicity through TGF‐β1‐p38 MAPK Signaling Pathway

Figure 1. Description of Sophora japonica L. and RUT. (a) Gross morphology of Sophora japonica L. (b) Chemical structure of RUT. RUT, rutinum.

Figure 2. Posttreatment cell viability assessment on MTT assay. #P < 0.05 versus control; ∗P < 0.05 versus THP; △P < 0.05 versus DZR + THP. aP < 0.05 versus RUT (50 μM) + THP; bP < 0.05 versus SB203580 + THP; cP < 0.05 versus RUT + SB203580 + THP. DZR, dexrazoxane; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; THP, pirarubicin.

Figure 3. Intracellular ROS concentration. After drug treatment, intracellular ROS concentration was assessed on DCFH-DA probing. (a) Representative images of DCFH-DA staining (original magnification ×200); (b) the average fluorescence intensity was quantified. #P < 0.05 versus control; ∗P < 0.05 versus THP; △P < 0.05 versus DZR + THP; aP < 0.05 versus RUT (50 μM) + THP; bP < 0.05 versus SB203580 + THP; cP < 0.05 versus RUT + SB203580 + THP. ROS, reactive oxygen species; DCFH-DA, dichlorodihydrofluorescein diacetate; THP, pirarubicin; DZR, dexrazoxane; RUT, rutinum; MFI, mean fluorescent intensity; DCF, 2′,7′-dichlorofluorescein.

Figure 4. Determination of cell apoptosis by Hoechst 33258 staining. (a) Representative images of Hoechst 33258 staining (original magnification ×200); (b) average percentage of apoptotic cells. #P < 0.05 versus control; ∗P < 0.05 versus THP; △P < 0.05 versus DZR + THP; aP < 0.05 versus RUT (50 μM) + THP; bP < 0.05 versus SB203580 + THP; cP < 0.05 versus RUT + SB203580 + THP. THP, pirarubicin; DZR, dexrazoxane; RUT, rutinum.

Figure 5. Determination of cell apoptosis on Annexin V/PI staining followed by flow cytometric analysis. (a) Representative data of flow cytometric analysis; (b) average percentage of early apoptotic cells (Annexin V+/PI−). #P < 0.05 versus control; ∗P < 0.05 versus THP; △P < 0.05 versus DZR + THP; aP < 0.05 versus RUT (50 μM) + THP; bP < 0.05 versus SB203580 + THP; cP < 0.05 versus RUT + SB203580 + THP.

Figure 6. Western blot analysis of protein expression. (a) Representative data of Western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal control. Semiquantitative analysis of TGF-β1, p-p38 MAPK, cleaved Caspase-9, Caspase-7, and Caspase-3 levels were presented in (b–f). The relative expression of p-p38 MAPK was normalized to that of total p38 MAPK and others were normalized to that of GAPDH. #P < 0.05 versus control; ∗P < 0.05 versus THP; △P < 0.05 versus DZR + THP. 1 = control, 2 = THP group, 3 = DZR + THP group, 4 = RUT + THP group, 5 = SB203580 + THP group, and 6 = SB203580 + THP + RUT group. DZR, dexrazoxane; MAPK, mitogen-activated protein kinase; RUT, rutinum; THP, pirarubicin; TGF-β1, transforming growth factor-β1.