Contributed equally
Icariin, the major active component isolated from plants of the Epimedium family, has been reported to have potential protective effects on the cardiovascular system. However, it is not known whether icariin has a direct effect on angiotensin II (Ang II)-induced cardiomyocyte enlargement and apoptosis. In the present study, embryonic rat heart-derived H9c2 cells were stimulated by Ang II, with or without icariin administration. Icariin treatment was found to attenuate the Ang II-induced increase in mRNA expression levels of hypertrophic markers, including atrial natriuretic peptide and B-type natriuretic peptide, in a concentration-dependent manner. The cell surface area of Ang II-treated H9c2 cells also decreased with icariin administration. Furthermore, icariin repressed Ang II-induced cell apoptosis and protein expression levels of Bax and cleaved-caspase 3, while the expression of Bcl-2 was increased by icariin. In addition, 2′,7′-dichlorofluorescein diacetate incubation revealed that icariin inhibited the production of intracellular reactive oxygen species (ROS), which were stimulated by Ang II. Phosphorylation of c-Jun N-terminal kinase (JNK) and p38 in Ang II-treated H9c2 cells was blocked by icariin. Therefore, the results of the present study indicated that icariin protected H9c2 cardiomyocytes from Ang II-induced hypertrophy and apoptosis by inhibiting the ROS-dependent JNK and p38 pathways.
Cardiac hypertrophy occurs when the heart endures overload or injury. Although hypertrophy is an adaptive process that initially maintains cardiac output, sustained hypertrophy ultimately leads to heart failure, which is the leading cause of morbidity and mortality worldwide (
Icariin (C33H40O15; molecular weight, 676.66), a prenylated flavonol glycoside, is the major active component isolated from plants of the Epimedium family (
Ang II functions as a significant hormonal mediator in cardiac hypertrophy that can induce a direct injury on cardiomyocytes. Reactive oxygen species (ROS)-dependent activation of the c-Jun N-terminal kinase (JNK) and p38 pathways has been shown to play a critical role in the effect Ang II exhibits on cardiomyocytes (
Icariin (≥94% purity as determined by high performance liquid chromatography analysis), Ang II and 2′,7′-dichlorofluorescein diacetate (DCFH-DA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium: Nutrient mixture F-12 (DMEM/F12), fetal bovine serum (FBS), trypsin, penicillin and streptomycin were purchased from Gibco-BRL (Carlsbad, CA, USA). TRIzol, Alexa Fluor® 488 goat anti-mouse IgG and SlowFade Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). A Transcriptor First Strand cDNA synthesis kit and Light Cycler 480 SYBR Green 1 Master Mix were purchased from Roche Diagnostics (Basel, Switzerland). Antibodies against α-actinin and an ApopTag® Plus Fluorescein In Situ Apoptosis detection kit were purchased from Millipore Corporation (Billerica, MA, USA). Primary antibodies were purchased from Cell Signaling Technology, Inc. (Beverley, MA, USA) and IRDye 800CW conjugated secondary antibodies were obtained from LI-COR Biosciences (Lincoln, NE, USA).
The H9c2 embryonic rat heart-derived cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Icariin was dissolved in dimethyl sulfoxide at a concentration of 10 mmol/l for storage. Cells were cultured in DMEM/F12 1:1 medium, supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin, in a humidified incubator with an atmosphere of 5% CO2 at 37°C. Cells were seeded at a density of 1×106 cells per well into six-well culture plates for mRNA extraction, 5×105 cells per well into six-well culture plates for cell surface area (CSA) and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) analysis, 5×103 cells per well in 96-well plates for ROS detection and 1×107 cells per well into 100 mm culture dishes for protein extraction. The cells were cultured in serum-free DMEM/F12 1:1 medium for 24 h and pretreated with icariin for 1 h prior to stimulation with Ang II.
Cell viability was analyzed using the Cell Counting Kit-8 (CCK-8) assay. Following icariin treatment for 48 h, 10 μl CCK-8 solution was added to each well of the 96-well plate and then incubated for an additional 4 h. Absorbance was measured at 450 nm using a microplate reader (Synergy HT; BioTek, Winooski, VT, USA). The percentage of cell viability was calculated according to the following formula: Cell viability (%) = optical density (OD) of the treatment group/OD of the control group × 100%.
To detect the mRNA expression levels of hypertrophic markers, including atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), qPCR was performed as described previously (
To assess CSA, cells were stained by immunofluorescence for cardiac α-actinin (
Apoptotic nuclei were labeled using TUNEL staining with a ApopTag® Plus Fluorescein In Situ Apoptosis Detection kit, according to the manufacturer’s instructions (
Intracellular ROS generation was determined using DCFH-DA, which becomes fluorescent on oxidation to DCF by H2O2 produced within cells. Following Ang II or/and Icariin treatments, H9c2 cells were washed twice and incubated with 5 μM DCFH-DA solution in serum-free medium at 37°C for 30 min in the dark. Data were then collected using a fluorescent reader (Synergy HT; BioTek) at excitation/emission wavelengths of 485/530 nm. A fluorescent microscope was also used to evaluate the DCF fluorescence of the cells on the coverslips.
Western blotting was performed as described previously (
Data are presented as the mean ± SEM and analyzed using a statistical software (SPSS 16.0; SPSS Inc., Chicago, IL, USA). Differences among the groups were determined by two-way analysis of variance followed by Tukey’s post hoc test. A comparison between the control and all the treatment groups was performed using the unpaired Student’s t-test. P<0.05 was considered to indicate a statistically significant difference.
The potential cytotoxicity of icariin was analyzed using a CCK-8 assay. H9c2 cells were incubated with various concentrations of icariin (0.1, 1, 5 or 10 μM) for 48 h. Cell viability in icariin-treated cells exhibited no significant differences when compared with the control cells, indicating that icariin at a concentration of 0.1, 1, 5 or 10 μM did not possess any cytotoxicity in H9c2 cells (
The effect of icariin at various concentrations (0.1, 1, 5 or 10 μM) on the induction of ANP and BNP in response to Ang II was determined. Stimulation with Ang II for 24 h markedly increased the mRNA expression levels of ANP and BNP in H9c2 cells and icariin treatment markedly attenuated this increase in a concentration-dependent manner (
CSAs of H9c2 cells were determined by α-actinin staining to further evaluate the antihypertrophic effect of icariin. Ang II stimulation for 48 h resulted in a significant increase in the CSAs of H9c2 cells. However, icariin treatment markedly attenuated the increase, indicating that Ang II-induced enlargement of H9c2 cells was suppressed by icariin (
To investigate the role of icariin in Ang II-induced apoptosis of H9c2 cells, TUNEL staining was used to identify the apoptotic nuclei. A marked increase in the number of TUNEL-positive nuclei was observed in cells that had been incubated with Ang II, and icariin treatment markedly reduced Ang II-induced cell apoptosis (
Fluorescence intensity in cells following incubation with DCFH-DA, exhibited by a fluorescent reader, revealed that Ang II increased the ROS content in a time-dependent manner. In addition, icariin treatment markedly blocked Ang II-induced ROS production at the indicated time points (
To further explore the mechanisms underlying the antihypertrophic and antiapoptotic effects of icariin in Ang II-treated H9c2 cells, western blotting was used to detect the phosphorylation levels of JNK and p38, which are key mediators of cardiac hypertrophy and apoptosis. Phosphorylated levels of JNK and p38 were shown to be markedly elevated by Ang II. However, icariin treatment inhibited the phosphorylation of JNK and p38 in response to Ang II at the indicated time points (
Icariin, the major active component isolated from plants of the Epimedium family, has been reported to exhibit potential protective effects on the cardiovascular system (
Enlargement and apoptotic loss of cardiomyocytes play critical roles in the transition from cardiac hypertrophy to heart failure (
Clinical and experimental studies have provided substantial evidence that ROS production, reflecting the status of oxidative stress, is enhanced in hypertrophic and failing hearts (
Increased ROS levels not only lead to the oxidation and damage of macromolecules, membranes and DNA, but also function as a secondary messengers in cellular signaling (
In conclusion, the current study has demonstrated a previously unknown effect of icariin on Ang II-induced cardiomyocyte hypertrophy and apoptosis through inhibiting the ROS-dependent JNK and p38 pathways. The results of the present study provide experimental evidence for the application of icariin in the treatment of cardiac hypertrophy.
The study was supported by grants from the National Natural Science Foundation of China (nos. 81300070 and 81270303) and the Fundamental Research Funds for the Central Universities of China (no. 2012302020212).
Effect of icariin on cell viability, as determined using the CCK-8 assay. Treatment with the indicated concentrations of icariin for 48 h did not cause any significant change in cell viability when compared with the control group. CCK-8, cell counting kit-8.
Effect of icariin on the mRNA expression levels of ANP and BNP. (A) Icariin decreased Ang II-induced mRNA expression levels of ANP and BNP in a concentration-dependent manner. *P<0.05, vs. control; #P<0.05, vs. Ang II-treated cells. (B) Icariin (10 μM) decreased Ang II (1 μM)-induced mRNA expression levels of ANP and BNP at the indicated time points. *P<0.05, vs. cells at 0 time; #P<0.05, vs. Ang II-treated cells at 0 time. Ang II, angiotensin II; ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide.
Effect of icariin on CSA. (A) Representative images and (B) quantitative results demonstrating that 10 μM icariin inhibited Ang II (1 μM; 48 h)-induced enlargement of H9c2 cells. *P<0.05, vs. control; #P<0.05, vs. Ang II-treated cells. CSA, cell surface area; Ang II, angiotensin II.
Effect of icariin on apoptosis. (A) Representative images and (B) quantitative results demonstrating that 10 μM icariin attenuated cell apoptosis following stimulation with Ang II for 48 h, as shown by TUNEL staining. (C) Representative blots and (D) quantitative results demonstrating that 10 μM icariin decreased the protein expression levels of Bax and cleaved-caspase 3, but increases the expression levels of Bcl-2 in response to Ang II stimulation. *P<0.05, vs. control; #P<0.05, vs. Ang II-treated cells. Ang II, angiotensin II; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling.
Effect of icariin on ROS production. (A) Icariin (10 μM) blocked 1 μM Ang II-induced ROS production at the indicated time points, as detected by a fluorescent reader. (B) Fluorescent microscope images demonstrating that 10 μM icariin attenuated DCF-derived fluorescence in cells treated with 1 μM Ang II for 2 h. *P<0.05, vs. cells at 0 time; #P<0.05, vs. Ang II-treated cells at 0 time. ROS, reactive oxygen species; Ang II, angiotensin II; DCF, 2′7′-dichlorofluorescein.
Effect of icariin on the activation of JNK and p38 pathways. (A) Representative blots and (B) quantitative results demonstrating that icariin decreased the phosphorylated levels of JNK and p38 in response to Ang II at the indicated time points. *P<0.05, vs. cells at 0 time; #P<0.05, vs. Ang II-treated cells at 0 time. Ang II, angiotensin II; JNK, c-Jun N-terminal kinase.