Figure 1.
Molecular structure of isorhamnetin.
Figure 2.
Effects of isorhamnetin on Dox-induced myocardial injury in vivo.
Rats were intraperitoneally (i.p.) treated with vehicle or Dox (3 mg/kg every other day for a cumulative dose of 9 mg/kg) with isorhamnetin pretreatment (5 mg/kg i.p. before Dox administration). After 28 days, the relative heart weight index (heart weight–body weight ratio, g/g) was measured (A). The effects of isorhamnetin treatment on LDH release (B), AST (C), and CK (D) levels were detected according to the manufacturer’s instructions. The effects of isorhamnetin on histological changes in the rat hearts was measured by HE staining (E); Cont, vehicle treatment; Iso, isorhamnetin treatment; Dox, doxorubicin treatment; Dox+Iso, isorhamnetin and doxorubicin co-treatment. Dotted arrow indicated infiltrated neutrophilic granulocyte, and solid arrow did cytoplasmic vacuolation. Results are represented as means ± SD (n = 9 per group). #P<0.05 vs. Cont; *P<0.05 vs. Dox-treated group.
Figure 3.
Effects of Dox and isorhamnetin on cell viability and LDH release in vitro.
H9c2 cardiomyocytes were treated for 6, 12, 24 and 36 h with different concentrations of Dox, and cell viability was determined by CCK8 assay (A). H9c2 cardiomyocytes were treated with different concentrations of isorhamnetin for 12 h, and cell viability was expressed as the relative percentage of control group (B). Control and isorhamnetin treated cells were further exposed to 1 µM Dox for 36 h; the cell viability (C) and LDH release (D) were measured. Results are represented as means ± SD from three independent experiments. #P<0.05 vs. Cont; *P<0.05 vs. Dox-treated group.
Figure 4.
Effects of Dox and isorhamnetin on cell viability and LDH release in vitro.Effects of Dox and isorhamnetin on antioxidant capacity in vivo and in vitro.
H9c2 cardiomyocytes were treated with vehicle or Dox for 0 h to 36 h and ROS fluorescence was detected on the automatic microplate reader at different times (A). The effects of isorhamnetin on the ROS production were also measured with the automatic microplate reader and visualized by fluorescence microscopy (B and C). Effects of isorhamnetin on activities of SOD, MDA, CAT, and GSH-Px in H9c2 cardiomyocytes (D), and rat heart tissues (E) were measured by corresponding detection kits. Cont, vehicle treatment; Iso, isorhamnetin treatment; Dox, doxorubicin treatment; Dox+Iso, isorhamnetin and doxorubicin co-treatment. Results are represented as means ± SD (n = 3–9 per group). #P<0.05 vs. Cont; *P<0.05 vs. Dox-treated group.
Figure 5.
Effects of Dox and isorhamnetin on apoptosis in vivo and in vitro.
H9c2 cardiomyocytes were incubated during increasing times with or without Dox (1 µM) and caspase-3 activity (units per microgram of protein) was assayed as described in Materials and Methods (A). Control and isorhamnetin-treated cells were further exposed to 1 µM of Dox for 36 h; caspase-3 fluorescence and Hoechst 33342 and propidium iodide (PI) double staining were used for the qualitative and quantitative analyses of the apoptotic cells (B and C). The internucleosomal DNA fragmentation was also determined by TUNEL assay in vitro and in vivo (D, scale bar). The TUNEL apoptotic index was determined by calculating the ratio of TUNEL-positive cells to total cells (E and F). Rat heart sections were also analyzed by immunohistochemistry with an antibody reactive to caspase-3 (G). Cont, vehicle treatment; Iso, isorhamnetin treatment; Dox, doxorubicin treatment; Dox+Iso, isorhamnetin and doxorubicin co-treatment. Results are represented as means ± SD (n = 3–9 per group). #P<0.05 vs. Cont; *P<0.05 vs. Dox-treated cells.
Figure 6.
Effects of Dox and isorhamnetin on mitochondrial apoptotic pathway in vivo and in vitro.
Mitochondrial injuries in rat heart tissues were observed by transmission electron microscopy (A). H9c2 cardiomyocytes stained with JC-1 dye were visualized by fluorescence microscopy (B). Cytochrome c (cyt c), caspase-9/3, and PARP in rat heart tissues and H9c2 cardiomyocytes were determined by western blot analysis (C). Cont, vehicle treatment; Iso, isorhamnetin treatment; Dox, doxorubicin treatment; Dox+Iso, isorhamnetin and doxorubicin co-treatment.
Figure 7.
Effects of Dox and isorhamnetin on expression of Bcl-2 family proteins in vivo and in vitro.
The expression levels of Bcl-2, Bax, and P53 were detected using an immunoblotting assay in vitro (A) and in vivo (B) and expressed as the fold changes over the control (C and D). Cont, vehicle treatment; Iso, isorhamnetin treatment; Dox, doxorubicin treatment; Dox+Iso, isorhamnetin and doxorubicin co-treatment. Results are represented as means ± SD from three independent experiments. #P<0.05 vs. Cont; *P<0.05 vs. Dox-treated group.
Figure 8.
Effects of Dox and isorhamnetin on activiation of MAPK in vitro.
H9c2 cardiomyocytes exposed to 1 µM of Dox for the indicated times (0 h to 36 h) were subjected to western blot analysis (A). H9c2 cardiomyocytes were treated with vehicle or Dox (1 µM, 36 h) with or without isorhamnetin (12.5 µg/ml) for 12 h prior to Dox exposure. The expression levels of ERK1/2, p38, and JNK were detected by immunoblotting assay. Cont, vehicle treatment; Iso, isorhamnetin treatment; Dox, doxorubicin treatment; Dox+Iso, doxorubicin and isorhamnetin co-treatment.
Figure 9.
Effects of isorhamnetin on the antitumor ability of Dox in vitro.
A series of cancer cell lines, including MCF-7 (a), HepG2 (b), and Hep2 (c) cells were treated with Dox (1 µM ) and isorhamnetin (6.25 µg/ml) alone or co-incubation for 36 h. The cell viability was measured by CCK8 assay (A). The cells stained with Annexin V-FITC/PI measured by Flow cytometry (B) and statistical analysis of the flow cytometry was shown (C). Cont, vehicle treatment; Iso, isorhamnetin treatment; Dox, doxorubicin treatment; Dox+Iso, doxorubicin and isorhamnetin co-treatment. Results are represented as means ± SD from three independent experiments. *P<0.05 vs. Cont; #P<0.05 vs. Dox-treated group.