Mechanism for effect of tanshinone IIA on alleviating cardiomyocyte injury induced by oxygen glucose deprivation

Abstract

Objectives: Tanshinone IIA has a wide range of myocardial protective effects. AK003290 is a long noncoding RNA (lncRNA) that is highly expressed in myocardial tissue, and its expression is down-regulated when myocardial injury occurs. This study aims to explore the mechanism for tanshinone IIA in alleviating myocardial cell damage induced by oxygen glucose deprivation (OGD).

Methods: OGD model was established in rat H9C2 cardiomyocytes. siRNA was transfected to reduce AK003290 expression. H9C2 cells were divided into 6 groups: A control group, a tanshinone IIA (TAN) group, an OGD group, a tanshinone IIA+OGD (TAN+OGD) group, a scrambled siRNA transfection+tanshinone IIA+OGD (scrambled siRNA+TAN+OGD) group, and a AK003290 siRNA transfection+tanshinone IIA+OGD (AK003290 siRNA+TAN+OGD) group. H9C2 cells in the TAN group were treated with 40 μmol/L tanshinone IIA for 12 h. The TAN+OGD group was treated with 40 μmol/L tanshinone IIA for 12 h, followed by OGD treatment for 12 h. The scrambled siRNA+TAN+OGD group and AK003290 siRNA+TAN+OGD group were transfected with the scrambled siRNA or AK003290 siRNA. Twenty-four hours later, the cells were treated with tanshinone IIA and OGD. Real-time RT-PCR was used to detect the expression of AK003290. Spectrophotometry was used to detect the content of lactate dehydrogenase (LDH) in cell culture medium to reflect LDH leakage rate, and enzyme-linked immunosorbent assay (ELISA) was used to detect the content of interleukin-1β (IL-1β) and interleukin-18 (IL-18). Western blotting was used to detect the protein expression of phospho-nuclear factor- κB (p-NF-κB).

Results: Compared with the control group, the leakage rate of LDH, the content of IL-1β and IL-18 in culture medium, and the protein expression level of p-NF-κB were increased in the OGD group (P<0.01 or P<0.001). Compared with the OGD group, the leakage rate of LDH, the content of IL-1β and IL-18 in culture medium, and the protein expression level of p-NF-κB were decreased in the TAN+OGD group (P<0.05 or P<0.01). Compared with the control group, the AK003290 expression was increased in the TAN group (P<0.01) and it was decreased in the OGD group (P<0.05). Compared with the OGD group, the AK003290 expression was increased in the TAN+OGD group (P<0.05). Compared with the scrambled siRNA+TAN+OGD group, the leakage rate of LDH, the content of IL-1β and IL-18 in culture medium, and the protein expression level of p-NF-κB were increased in the AK003290 siRNA+TAN+OGD group (P<0.05 or P<0.01).

Conclusions: Tanshinone IIA inhibits NF-κB activity and attenuates OGD-induced inflammatory injury of cardiomyocytes through up-regulating AK003290.

Keywords: AK003290; cardiomyocyte injury; inflammatory; long noncoding RNA; nuclear factor- κB; oxygen glucose deprivation; tanshinone IIA.

图1

丹参酮IIA和/或OGD处理对LDH漏出率(A)及细胞培养液中IL-1β(B)和IL-18(C)含量的影响 Figure 1 Effect of treatment with tanshinone IIA and/or OGD on the LDH leakage rate (A), and content of IL-1β (B) and IL-18 (C) in cell culture medium Pretreatment of H9C2 cells with 40 μmol/L tanshinone IIA for 12 h inhibited the increased leakage of LDH and release of inflammatory cytokines in H9C2 cells induced by OGD treatment. Data are represented as the means±standard deviation, n=6. **P<0.01, ***P<0.001 vs the control group; †P<0.05, ††P<0.01 vs the OGD group. LDH: Lactate dehydrogenase; IL-1β: Interleukin-1β; IL-18: Interleukin-18; OGD: Oxygen glucose deprivation.

图2

丹参酮IIA和/或OGD处理对p-NF-κB蛋白质表达水平的影响 Figure 2 Effect of treatment with tanshinone IIA and/or OGD on the protein expression level of p-NF-κB The protein expression level of p-NF-κB in H9C2 cells was detected by Western blotting. Pretreatment of H9C2 cells with 40 μmol/L tanshinone IIA for 12 hours inhibited the increased protein expression level of p-NF-κB in H9C2 cells induced by OGD treatment. Data are represented as the means±standard deviation, n=3. **P<0.01 vs the control group; †P<0.05 vs the OGD group. p-NF-κB: Phospho-nuclear factor-κB; OGD: Oxygen glucose deprivation.

图3

丹参酮IIA和/或OGD处理对 AK003290 表达水平的影响 Figure 3 Effect of treatment with tanshinone IIA and/or OGD on the expression level of AK003290 The expression level of AK003290 in H9C2 cells was detected by real-time RT-PCR. Pretreatment of H9C2 cells with 40 μmol/L tanshinone IIA for 12 h inhibited the decreased expression of AK003290 in H9C2 cells induced by OGD treatment. Data are represented as the means±standard deviation, n=6. *P<0.05, **P<0.01 vs the control group; †P<0.05 vs the OGD group. OGD: Oxygen glucose deprivation.

图4

敲低 AK003290 基因的表达(A)对LDH漏出率(B)及细胞培养液中IL-1β(C)和IL-18(D)含量的影响 Figure 4 Effect of knocking down the expression of AK003290 gene (A) on the LDH leakage rate (B) and content of IL-1β (C) and IL-18 (D) in the cell culture medium H9C2 cells transfected with AK003290 siRNA were pretreated with 40 μmol/L tanshinone IIA for 12 h, and then treated with OGD for 12 h. Knocking down the expression of AK003290 gene can increase the leakage rate of LDH and the release of inflammatory cytokines in H9C2 cells induced by OGD treatment. Data are represented as the means±standard deviation, n=6. ***P<0.001 vs the scrambled siRNA group, †P<0.05 vs the scrambled siRNA+TAN+OGD group. LDH: Lactate dehydrogenase; IL-1β: Interleukin-1β; IL-18: Interleukin-18; OGD: Oxygen glucose deprivation.

图5

敲低 AK003290 基因的表达对p-NF-κB蛋白质表达水平的影响 Figure 5 Effect of knocking down the expression of AK003290 gene on the protein expression level of of p-NF-κB The protein expression level of p-NF-κB in H9C2 cells was detected by Western blotting. H9C2 cells transfected with AK003290 siRNA were pretreated with 40 μmol/L tanshinone IIA for 12 h, and then treated with OGD for 12 h. Knocking down the expression of AK003290 gene can increase the protein expression level of p-NF-κB. Data are represented as the means±standard deviation, n=3. **P<0.01 vs the scrambled siRNA+TAN+OGD group. p-NF-κB: Phospho-nuclear factor-κB; OGD: Oxygen glucose deprivation.