Shengxian decoction decreases doxorubicin‑induced cardiac apoptosis by regulating the TREM1/NF‑κB signaling pathway

Abstract

Shengxian decoction (SXT) is a traditional Chinese medicine that is clinically used for treating cardiovascular diseases. It is known for its beneficial effect on cardiomyocyte injuries, some of which can be induced by anticancer agents including doxorubicin (DOX). To determine the molecular mechanisms involved in the cardioprotective effects of SXT, DOX‑induced H9c2 cells were analyzed for apoptosis and expression levels of apoptosis biomarkers. Cell viability and apoptosis were measured by CCK‑8 and flow cytometry. Triggering receptors expressed on myeloid cells 1 (TREM1), cleaved caspase‑3, survivin and NF‑κBp65 expression levels were measured by reverse transcription‑quantitative PCR and/or western blotting. A total of 30 adult male Sprague‑Dawley rats were randomly allocated into five groups (n=6 each); control group receiving 0.9% saline, 1 DOX group receiving 2.5 mg/kg of DOX and 3 DOX + SXT groups, receiving a DOX dose equivalent to the DOX‑only group and either 0.4, 0.8 or 1.6 g/kg of SXT. It was found that DOX increased apoptosis and NF‑κB activation of H9c2 cells by increasing TREM1 expression and that SXT inhibited apoptosis and NF‑κB activation of H9c2 cells induced by DOX or Trem1 overexpression. SXT also significantly reversed DOX‑induced cardiotoxicity in rats. The results suggested that the protective effects of SXT against DOX‑induced apoptosis may be attributed to its downregulation of TREM1.

Keywords: doxorubicin; Shengxian decoction; apoptosis; triggering receptors expressed on myeloid cells 1; NF‑κB.

Figure 1.

DOX treatment increased the expression of TREM1 in H9c2 cells. (A) Confirmation of suppressive effects of DOX on H9c2 cell proliferation, detected by CCK-8 assay. (B and C) TREM1 expression in response to different DOX concentrations was measured by (B) reverse transcription-quantitative PCR and (C) western blotting. All data are expressed as mean ± standard deviation of triplicate dependent experiments. *P<0.05 and ***P<0.001 vs. no-treatment group. DOX, doxorubicin; TREM1, triggering receptors expressed on myeloid cells 1.

Figure 2.

SXT inhibited the increased apoptosis of H9c2 cells induced by DOX. (A) Effect of SXT on cell proliferation in DOX-exposed H9c2 cells. For experiment groups that were exposed to 2 µmol/l DOX for 12 h, the suppression decreased with an increase in SXT concentration and significant decrease can be found in cells pretreated with 0.5 mg/ml or more of SXT in comparison with no-treatment group. (B and C) Effect of SXT on apoptosis level of DOX-exposed H9c2 cells, detected by flow cytometer. Shown in the column is the sum of apoptotic cells (early and late stage) and necrotic cells in percentages. (D) Western blotting analysis of TREM1, cleaved caspase-3, survivin and NF-κB. All data are expressed as mean ± standard deviation of triplicate dependent experiments. ***P<0.001 vs. control group; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. DOX group. SXT, Shengxian decoction; DOX, doxorubicin; TREM1, triggering receptors expressed on myeloid cells 1.

Figure 3.

DOX increases apoptosis of H9c2 cells by increasing TREM1. TREM1 expression in H9c2 cells with Trem1 siRNA silencing and overexpression was measured by (A) reverse transcription-quantitative PCR and (B and C) western blotting. (D and E) Silencing of Trem1 can decrease apoptosis level of DOX-exposed H9c2 cells. (F and G) Protein profile of TREM1, cleaved caspase-3, survivin and NF-κB in H9c2 cells treated as indicated. All data are expressed as mean ± standard deviation of triplicate dependent experiments. **P<0.01 and ***P<0.001 vs. control group; ^##P<0.01, ^###P<0.001 vs. DOX + siNC group. DOX, doxorubicin; TREM1, triggering receptors expressed on myeloid cells 1; si, small interfering.

Figure 4.

STX inhibits apoptosis of H9c2 cells induced by TREM1. (A and B) Increased apoptosis level in cells with TREM1 overexpression compared with control treated with an empty vector and when regulated by 1 mg/ml SXT. (C) Protein profile of TREM1, cleaved caspase-3, survivin and NF-κB in H9c2 cells treated as indicated. All data are expressed as mean ± standard deviation of triplicate dependent experiments. ***P<0.001 vs. vector group; ^###P<0.001 vs. oeTrem1 + SXT group. SXT, Shengxian decoction; TREM1, triggering receptors expressed on myeloid cells 1; oe, overexpression.

Figure 5.

Protective effect of STX in DOX-induced rats. (A) Micrographs of H&E- and TUNEL-stained cardiomyocytes from experiment rats of control and DOX-induced heart failure model group. Scale bar=100 µm. (B) Histopathological score of the different groups. (C) Percentage of TUNEL-positive cells. (D) Quantification of BNP in blood measured by ELISA. (E) Confirmation of changes in protein levels in rats with different treatments. All data are expressed as mean ± standard deviation of triplicate dependent experiments. ***P<0.001 vs. control group; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs. DOX group. SXT, Shengxian decoction; DOX, doxorubicin; H&E, haemotoxylin and eosin; BNP, brain natriuretic peptide.