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. 2017 Oct;14(4):3523-3532.
doi: 10.3892/etm.2017.4964. Epub 2017 Aug 18.

"VSports最新版本" Suxiao Jiuxin Pill protects cardiomyocytes against mitochondrial injury and alters gene expression during ischemic injury

Xiaofen Ruan  1 , Tiejun Chen  1 , Xiaolong Wang  1 , Yiping Li  1
Affiliations

Suxiao Jiuxin Pill protects cardiomyocytes against mitochondrial injury and alters gene expression during ischemic injury

Xiaofen Ruan et al. Exp Ther Med. 2017 Oct.

Abstract

Suxiao Jiuxin Pill (SX), a traditional Chinese medicine compound consisting primarily of tetramethylpyrazine and borneol, has been reported to protect against ischemic heart disease. However, the effects of SX on mitochondrial injury and gene expression in various signaling pathways are unclear. The aim of the present study was to investigate the effects of SX on mitochondrial injury and to screen the expression of genes potentially altered by SX using a cell culture model of ischemic injury. Simulated ischemia was established by culturing HL-1 cardiomyocytes in Dulbecco's modified Eagle medium without glucose or serum in a hypoxic chamber containing 95% N2 and 5% CO2 for 24 h. HL-1 cardiomyocytes were divided into 3 groups: Control, ischemic injury and ischemic injury + SX (100 µg/ml; n=3 wells/group). Mitochondrial membrane potential was detected by staining with JC-1 dye. The mRNA expression levels of adenylyl cyclase (Adcy) 1-9, adrenoceptor β1, Akt1, ATPase Na+/K+ transporting subunit β2, calcium voltage-gated channel auxiliary subunit α2δ (Cacna2d)2, Cacna2d3, calcium channel voltage-dependent γ subunit 8, cytochrome C oxidase subunit 6A2 (Cox6a2), fibroblast growth factor receptor (Fgfr) 4, Fgf8, Fgf12, Gnas complex locus, glycogen synthase kinase 3β (Gsk3b), mitogen-activated protein kinase (Mapk)11-14, Mapk kinase kinase kinase 1 (Map4k1), Mas1, nitric oxide synthase 3 (Nos3), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (Pik3ca), phospholipase A2 group 4A, rap guanine nucleotide exchange factor 4 and ryanodine receptor 2 were detected using reverse transcription-quantitative polymerase chain reaction. The protein expression levels of phosphoinositide 3-kinase (PI3K), MAS-1 and phosphorylated-endothelial NOS were also examined by immunofluorescence staining. The decrease in mitochondrial membrane potential in the cell culture model of ischemic injury (P<0. 001) was significantly attenuated by SX treatment (P<0. 001). Furthermore, increases in the mRNA expression levels of Adcy2 (P<0. 05), 3 (P<0. 01) and 8 (P<0. 05) in the ischemic injury model were significantly attenuated by SX treatment (P<0. 01), and SX treatment significantly decreased the mRNA expression levels of Adcy1 (P<0. 01) and 6 (P<0. 05) in ischemic cells. Decreases in the mRNA expression levels of Cox6a2 (P<0. 001), Gsk3b (P<0. 01) and Pik3ca (P<0. 001) in the ischemic injury model were also significantly attenuated by SX treatment (P<0. 05, P<0. 01 and P<0. 001, respectively). In addition, the decrease in the protein expression of PI3K (P<0. 001) was significantly attenuated by SX treatment (P<0. 001) VSports手机版. The present findings indicate that SX may protect cardiomyocytes against mitochondrial injury and attenuate alterations in the gene expression of Adcy2, 3 and 8, Cox6a2, Gsk3b and Pik3ca during ischemic injury. .

Keywords: HL-1 cells; Suxiao Jiuxin Pill; ischemic injury; mRNA; mitochondria V体育安卓版. .

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Figures

Figure 1.
Figure 1.
Determination of the optimal dosage of SX. HL-1 cardiomyocytes were divided into 3 groups: Control, ischemic injury, and ischemic injury + SX. Induced ischemia was established by culturing HL-1 cardiomyocytes in glucose- and serum-free Dulbecco's modified Eagle's medium in a hypoxic chamber with 95% N2 and 5% CO2 for 24 h. Cells in the control group were cultured in Dulbecco's modified Eagle medium containing glucose and serum in a normoxic chamber with 5% CO2 for 24 h. To determine the optimal SX dosage, SX (10, 50, 100, 200 and 400 µg/ml, respectively) was added to cell culture medium at the start of hypoxia. Cell morphology was observed using light microscopy. The growth of HL-1 cardiomyocytes was optimal following treatment with 100 µg/ml SX, which indicated that 100 µg/ml SX exerted the greatest protective effects against ischemic injury. Therefore, 100 µg/ml SX was used for subsequent experiments. Magnification, ×100; scale bar=100 µm. SX, Suxiao Jiuxin Pill.
Figure 2.
Figure 2.
SX treatment attenuated the decrease in mitochondrial membrane potential induced by ischemic injury. HL-1 cardiomyocytes in 3 experimental groups were stained with JC-1 dye (red) and observed under a fluorescence microscope (magnification, ×100). Data were presented as the mean + standard error of the mean (n=3 wells/group). Red signal indicated positive (polarized) mitochondrial membrane potential. The mitochondrial membrane potential decreased significantly in the ischemic injury group when compared with the control group, and SX treatment significantly attenuated the decrease in mitochondrial membrane potential in ischemic HL-1 cardiomyocytes. ***P<0.001 vs. control group; ###P<0.001 vs. ischemic injury group. Scale bars=100 µm. SX, Suxiao Jiuxin Pill; IOD, integrated optical density.
Figure 3.
Figure 3.
SX treatment attenuated the increase in levels of Adcy2, 3 and 8 mRNA, and decreased the levels of Adcy1 and 6 mRNA during ischemic injury. mRNA expression in the 3 experimental groups was detected by reverse transcription-quantitative polymerase chain reaction. The levels of (A) Adcy2, (B) 3 and (C) 8 mRNA in HL-1 cardiomyocytes were significantly increased following ischemic injury, while the levels of (D) Adcy1 and (E) 6 mRNA did not differ significantly following ischemia. SX treatment attenuated the increases in Adcy2, 3 and 8 mRNA, and significantly decreased Adcy1 and 6 mRNA in the cell culture model of ischemic injury. Data were represented as the mean ± standard error of the mean (n=3 wells/group). *P<0.05 and **P<0.01 vs. control group; #P<0.05 and ##P<0.01 vs. ischemic injury group. SX, Suxiao Jiuxin Pill.
Figure 4.
Figure 4.
SX treatment attenuated the decrease in Cox6a2, Gsk3b and Pik3ca mRNA in the ischemic injury model. The levels of (A) Cox6a2, (B) Pik3ca and (C) Gsk3b mRNA were significantly decreased in the ischemic injury group when compared with the control group. The decreases in Cox6a2, Gsk3b and Pik3ca mRNA were significantly attenuated by SX treatment. Data were represented as the mean ± standard error of the mean (n=3 wells/group). **P<0.01 and ***P<0.001 vs. the control group; #P<0.05, ##P<0.01 and ###P<0.001 vs. the ischemic injury group. SX, Suxiao Jiuxin Pill.
Figure 5.
Figure 5.
Levels of Rapgef4, Mapk11, Mapk13, Mapk14 and Fgf8 mRNA were not altered by SX treatment in the ischemic injury model. The levels of (A) Rapgef4, (B) Mapk11, (C) Mapk13 and (D) Mapk14 mRNA were significantly increased, and the level of (E) Fgf8 mRNA was significantly decreased in the ischemic injury group when compared with the control group. SX treatment had no significant effect on the expression of these genes in ischemic cardiomyocytes. Data were represented as the mean ± standard error of the mean (n=3 wells/group). *P<0.05 and **P<0.01 vs. the control group. SX, Suxiao Jiuxin Pill.
Figure 6.
Figure 6.
Reduced protein expression of PI3K was attenuated by SX treatment in the ischemic injury model. The levels of PI3K protein expression in the 3 experimental groups were examined by immunofluorescence staining (magnification, ×100). PI3K protein was stained with fluorescein isothiocyanate (green) and cells were counterstained with 4′,6-diamidino-2-phenylindole (blue). Data were represented as the mean + standard error of the mean (n=3 wells/group). The expression levels of PI3K were significantly decreased in the ischemic injury group compared with the control group, whereas SX treatment significantly restored the expression of PI3K to normal levels during ischemic injury. ***P<0.001 vs. the control group and ###P<0.001 vs. the ischemic injury group. Scale bars=100 µm. SX, Suxiao Jiuxin Pill; IOD, integrated optical density.
Figure 7.
Figure 7.
Protein expression of MAS1 and p-eNOS was similar in the 3 experimental groups. The protein expression levels of MAS-1 and p-eNOS in the 3 experimental groups were examined by immunofluorescence staining. The target proteins were stained with fluorescein isothiocyanate (green). No statistical differences in the expression levels of MAS1 and p-eNOS among the 3 groups were identified. Scale bar=100 µm. Data were represented as the mean ± standard error of the mean (n=3 wells/group). SX, Suxiao Jiuxin Pill; IOD, integrated optical density; eNOS, endothelial nitric oxide synthase; p-, phosphorylated.
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