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Icariin (ICA) has been implicated in certain biological and pathological processes, including myocardial ischemia/reperfusion (I/R) injury. The aim of the present study was to investigate the role of ICA in I/R-induced cardiomyocyte injury and the potential underlying mechanism. Cell proliferation and apoptosis of H9C2 cells was determined by cell counting kit-8 and flow cytometry assays. In addition, reactive oxygen species (ROS) production in H9C2 cells was measured by flow cytometry. Reverse transcription-quantitative polymerase chain reaction and western blot assay were performed to examine the expression levels of proteins, including HSP20, B-cell lymphoma 2 (Bcl-2), cytochrome complex (Cyt-c), apoptotic protease activating factor 1 (APAF1), caspase-9 andcaspase-3, and the phosphorylation of Akt (p-Akt) in H9C2 cells. The present results demonstrated that, compared with the control group, the I/R group demonstrated significantly reduced levels of HSP20 expression and cell proliferation, and increased apoptosis and ROS production in H9C2 cells. In parallel, the expression levels of Cyt-c, APAF1, caspase-9 and caspase-3 were significantly increased in the I/R group, although Bcl-2 and p-Akt/Akt expression levels were decreased. Furthermore, compared with the I/R group, ICA treatment and/or HSP20 overexpression significantly improved cardiac function, as evidenced by promoted cell proliferation and inhibited apoptosis of H9C2 cells. The current study indicates that ICA exerts a cardioprotective effect against I/R injury, which is associated with the upregulation of HSP20.
Myocardial ischemia refers to the heart with blood perfusion reduction, which occurs when the balance of myocardial blood supply and demand is disturbed (
Icariin (ICA) is a flavonoid extracted from
Increasing evidences supports a pivotal role for the small heat shock protein (HSP) family in multiple processes (
The present study was designed to further determine the cardioprotective effect of ICA on myocardial I/R injury and the molecular mechanism underlying HSP20.
H9C2 cells (Institute of Biochemistry and Cell Biology, Shanghai, China) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% 100X mycillin (Invitrogen; Thermo Fisher Scientific, Inc.), and incubated in 5% CO2 at 37°C overnight. Cells were digested and seeded into 96-well plates (3×103 cells/well). The I/R group was transferred into sugar and serum-free DMEM and incubated in 5% CO2 and 95% N2 at 37°C for 2 h, then transferred into normal DMEM and incubated in 5% CO2 at 37°C for 6 h. The control group was incubated in normal DMEM (Invitrogen; Thermo Fisher Scientific, Inc.) for 8 h at 37°C. In the drug-treated groups, ICA (Xian Xiao Cao Botanical Development Co., Ltd., Xian, China) at various concentrations (5, 10 and 15 µmol/l) was administered as a component of the perfusion medium 10 min before ischemia and every 8 h throughout reperfusion.
The HSP20 coding sequence (commercially unavailable; Sangon Biotech Co., Ltd., Shanghai, China) was cloned into the pAVsi 1.1 adenovirus vector and black pAVsi 1.1 adenovirus vector (both Sangon Biotech Co., Ltd.) served as a negative control. To generate a high-titer adenovirus, vectors encoding the HSP20 and the packaging plasmids were cotransfected into 293T cells (Institute of Biochemistry and Cell Biology) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. The packaging, purification and titration of adenovirus were performed as previously described (
H9C2 cells (1×103 cells/well) were plated in 96-well plates. Following ICA treatment for 48 h, 10% Cell Counting-kit 8 (CCK-8; CK04; Dojindo Molecular Technologies, Inc., Kumamoto, Japan) diluted in DMEM was mixed in each well for another 1 h. The absorption of each sample was measured at a wavelength of 450 nm using a Labsystems MK3 microplate reader (Thermo Fisher Scientific, Inc.) to detect cell viability according to the manufacturer's instruction.
Following transfection, H9C2 cells were detached using 0.25% trypsin and washed with 10% phosphate-buffered saline (PBS), followed by the centrifugation at 1,000 × g for 5 min at 37°C. Then, the cells were incubated with 10 µl Annexin V-fluorescein isothiocyanate (FITC) and 5 µl propidium iodide (PI) in the dark for 15 min at 4°C. Cell apoptotic rate was measured by Annexin V-FITC Apoptosis Detection kit (Beyotime Biotechnology, Shanghai, China) and the data was obtained using flow cytometer (BD Accuri C6 software version 1.0.264.21; BD Biosciences, Franklin Lakes, NJ, USA).
The intracellular ROS content was determined using a fluorescent probe, 2′,7′dichlorodihydrofluorescein-diacetate (DCFH-DA) followed by flow cytometry. After transfection, H9C2 cells were incubated with 10 µM DCFH-DA at 37°C for 20 min in the dark. Then, the plates were washed three times with PBS. The fluorescent probe DCFH-DA (Thermo Fisher Scientific, Inc.), which detected ROS production, was observed using a flow cytometer (BD Accuri C6 software version 1.0.264.21; BD Biosciences).
Total RNA was isolated from H9C2 cells using the TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) method, depurated with an RNAeasy kit (Invitrogen; Thermo Fisher Scientific, Inc.) and reversed to cDNA using the Prime-Script RT reagent kit (Takara Bio, Inc., Otsu, Japan). qPCR was performed using an ABI 7500 (Applied Biosystems; Thermo Fisher Scientific, Inc.) using SYBR Premix Ex Taq (Takara Bio, Inc.). The following primers were used: Sense, 5′-CTGTTTTGGTGAGGGGAAGG-3′ and antisense, 5′-CTGGGGAGAAATGGGATACG-3′ for HSP20; sense, 5′-GTCGGTGTGAACGGATTTG-3′ and antisense, 5′-TCCCATTCTCAGCCTTGAC-3′ for GAPDH. The HSP20 mRNA level was normalized against internal GAPDH mRNA. The relative quantification values for gene expression levels were calculated using 2−ΔΔCq method (
Upon termination of treatment, H9C2 cells were harvested and resuspended in ice-cold cell lysis solution and the homogenate was centrifuged at 400 × g for 15 min at 4°C. A bicinchoninic acid protein quantification kit (cat. no. 23225; Thermo Fisher Scientific, Inc.) was used to quantify the protein contents. Then, 30 µg protein was run on 12% SDS-PAGE gel and transferred to a nitrocellulose filter membrane (Merck KGaA, Darmstadt, Germany) electrophoretically. Blots were blocked with 5% skimmed milk at room temperature for 1 h, followed by incubation with anti-B-cell lymphoma 2 (Bcl-2; cat. no. sc-492; 1:150; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), caspase-3 (cat. no. ab2302; 1:200; Abcam, Cambridge, MA, USA), caspase-9 (cat. no. ab2013; 1:1,000; Abcam), cytochrome complex (Cyt-c; cat. no. ab13575; 1:1,000; Abcam), apoptotic protease activating factor 1 (APAF1; cat. no. 8969; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), Akt (cat. no. 2920; 1:2,000; Cell Signaling Technology, Inc.), p-AKT (cat. no. 4060; 1:2,000; Cell Signaling Technology, Inc.) and GAPDH (cat. no. 5174; 1:1,500; Cell Signaling Technology, Inc.) antibodies overnight at 4°C, and incubated with horse radish peroxidase-conjugated goat anti-mouse or anti-rabbit secondary antibody (cat nos. A0208 and A0216, respectively; 1:1,000; Beyotime Institute of Biotechnology, Shanghai, China) for 1 h at 37°C. Enhanced chemiluminescence (Thermo Scientific, Shanghai, China) was used to detect the blots visually and signals were quantified by densitometry (Quantity One software version 4.62; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Experiments were performed in triplicate and data were expressed as the mean ± standard deviation of the mean. Statistical significance was determined by unpaired two-tailed t-test and one-way analysis of variance followed by Tukey's post hoc test. Statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software, Inc., La Jolla, CA, USA) and P<0.05 was considered to indicate a statistically significant difference.
In order to investigate the possible mechanisms involved in the protective effect of ICA on cardiac cells against I/R injury, an
To clarify the effect of I/R on HSP20 expression levels
The effects of I/R on H9C2 cell proliferation and apoptosis were measured by CCK-8 and flow cytometry, respectively. As shown in
The effect of I/R on ROS production in H9C2 cells was measured by flow cytometry. As exhibited in
The effects of I/R on protein expression levels in H9C2 cells were analyzed by western blotting. As presented in
Cardiomyocyte apoptosis maybe a fundamental aspect of the myocardial process that initiates or aggravates heart failure. Consistent with the previously reported cardioprotective effects of ICA (
ICA, the major active component isolated from
PI3K/Akt is an intracellular signaling pathway, which is particularly important following ischemic insults. Activated Akt produces its anti-apoptotic effects via the phosphorylation of two categories of downstream substrates: The anti-apoptotic substrates (Bcl-2) and the pro-apoptotic substrates (caspase-9) (
In conclusion, the present study provides the first evidence, to the best of our knowledge, that ICA treatment protects the heart against I/R-induced apoptosis and ROS production, and this protective effect of ICA may be associated with an associated upregulation of HSP20. Further research is required to confirm the cardioprotective effect of ICA on I/R and to clarify the molecular mechanisms involving the Akt signaling pathway using LY294002, a PI3K-Akt signaling pathway inhibitor. The current data indicate that HSP20 presents as a potential therapeutic protein for ischemic diseases and additional studies are necessary.
Effects of ICA on I/R-induced injury in H9C2 cells. (A) Cell proliferation and (B and C) apoptosis in H9C2 cells following I/R treatment in the absence and presence of ICA treatment were evaluated by Cell Counting Kit-8 assay and flow cytometry, respectively. Data are presented as the mean ± standard deviation. ***P<0.001 vs. control. ΔΔP<0.01, ΔΔΔP<0.001 vs. I/R. ICA, icariin; I/R, ischemia/reperfusion; OD, optical density.
Expression levels of HSP20 in H9C2 cells following I/R treatment in the absence and presence of ICA treatment by (A) RT-qPCR and (B) western blot analysis. Expression levels of HSP20 in H9C2 cells following recombinant adenovirus transfection for 48 h by (C) RT-qPCR and (D) western blot analysis. Data are presented as the mean ± standard deviation. ***P<0.001 vs. control; ΔΔP<0.01, ΔΔΔP<0.001 vs. I/R. HSP, heat shock protein; I/R, ischemia/reperfusion; ICA, icariin; NC, negative control.
Effects of HSP20 overexpression on I/R-induced injury in H9C2 cells. (A) Cell proliferation and (B and C) apoptosis in H9C2 cells following I/R was evaluated by CCK-8 assay and flow cytometry. Data are presented as the mean ± standard deviation. ***P<0.001 vs. control; ΔP<0.05, ΔΔΔP<0.001 vs. I/R. HSP, heat shock protein; I/R, ischemia/reperfusion; NC, negative control; ICA, icariin.
Effects of HSP20 overexpression on I/R-induced ROS production in H9C2 cells. (A) ROS production was evaluated by flow cytometry in H9C2 cells with different treatments, including (B) control, (C) I/R, (D) I/R+ICA, (E) I/R+NC, (F) I/R+HSP20 and (G) I/R+HSP20+ICA. Data are presented as the mean ± standard deviation. ***P<0.001 vs. control; ΔΔP<0.01, ΔΔΔP<0.001 vs. I/R. HSP, heat shock protein; I/R, ischemia/reperfusion; ROS, reactive oxygen species; NC, negative control; ICA, icariin.
Effects of HSP20 overexpression on I/R-induced protein expression levels in H9C2 cells. Expression levels of p-Akt, Akt, Bcl-2, Cyt-c, APAF1, caspase-9 and caspase-3 in H9C2 cells following I/R treatment were analyzed by (A) western blotting and (B-D) quantified. Data are presented as the mean ± standard deviation. ***P<0.001 vs. control; ΔΔP<0.01, ΔΔΔP<0.001 vs. I/R. HSP, heat shock protein; I/R, ischemia/reperfusion; p, phosphorylated; Bcl-2, B-cell lymphoma 2; Cyt-c, cytochrome complex; APAF1, apoptotic protease activating factor 1; NC, negative control; ICA, icariin.