Comparative efficacy of sweated and non-sweated Salvia miltiorrhiza Bge. extracts on acute myocardial ischemia via regulating the PPARα/RXRα/NF-κB signaling pathway

Abstract

Salvia miltiorrhiza Bge. (S. miltiorrhiza) is a well-known traditional Chinese medicine for the treatment of cardiovascular diseases. The processing of S. miltiorrhiza requires the raw herbs to sweat first and then dry. The aim of this study was to investigate the anti-acute myocardial ischemia (AMI) of S. miltiorrhiza extracts (including tanshinones and phenolic acids) before and after sweating, and to further explore whether the "sweating" primary processing affected the efficacy of S. miltiorrhiza. The AMI animal model was established by subcutaneous injection of isoprenaline hydrochloride (ISO). After treatment, the cardiac function of rats was evaluated by electrocardiogram (ECG), biochemical, and histochemical analysis. Moreover, the regulation of S. miltiorrhiza extracts on the peroxisome proliferator-activated receptor α (PPARα)/retinoid X receptor α (RXRα)/nuclear transcription factor-kappa B (NF-κB) signaling pathway of rats was assessed by the Western blotting. The results showed that sweated and non-sweated S. miltiorrhiza extracts including tanshinones and phenolic acids significantly reduced ST-segment elevation in ECG and the myocardial infarction area in varying degrees. Meanwhile, sweated and non-sweated S. miltiorrhiza reversed the activities of aspartate transaminase (AST), lactic dehydrogenase (LDH), creatine kinase-MB (CK-MB), and superoxide dismutase (SOD), as well as the levels of interleukin-6 (IL-6), interleukin-10 (IL-10), and tumor necrosis factor-α (TNF-α) in AMI rats. Concurrently, the results of Western blotting revealed that S. miltiorrhiza extracts regulated the PPARα/RXRα/NF-κB signaling pathway to exert an anti-inflammatory effect. Most importantly, sweated S. miltiorrhiza tanshinones extracts are more effective than the non-sweated S. miltiorrhiza, and the anti-inflammatory efficacy of tanshinones extract was also better than that of phenolic acid extract. Although phenolic acid extracts before and after sweating were effective in anti-AMI, there was no significant difference between them. In conclusion, both tanshinones and phenolic acids extracts of sweated and non-sweated S. miltiorrhiza promote anti-oxidative stress and anti-inflammatory against AMI via regulating the PPARα/RXRα/NF-κB signaling pathway. Further, the comparations between sweated and non-sweated S. miltiorrhiza extracts indicate that sweated S. miltiorrhiza tanshinones extracts have better therapeutic effects on AMI.

Keywords: Acute myocardial ischemia; Anti-inflammatory; S. miltiorrhiza; Sweating.

Graphical abstract

Fig. 1

The extraction process of the NSPA, SSPA, SST, NST (A) and the illustration of the animal experiments protocol (B).

Fig. 2

The ECG images and ST-segment value in each group (n = 6) (A–G) The ECG of rats in the CMC-Na, model, IMST, NST-L, NST-H, SST-L, and SST-H groups. (H–L) The ECG of rats in the saline, NSPA-L, NSPA-H, SSPA-L and SSPA-H groups. (M − O) The value of ST-segment in groups. Compared with the saline group: ^####p < 0.001; compared with the AMI group: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05; the tanshinones extracts groups compared with the phenolic acid extracts groups: NS: no significant difference.

Fig. 3

The percentage of infarcted area in each group (n = 3) (A) TTC staining images of rat hearts in NST and SST groups. (B) TTC staining images of rat hearts in NSPA and SSPA groups. (C) The percentage of infarcted area in NST and SST groups. (D) The percentage of infarcted area in NSPA and SSPA groups. (E) Comparison of the percentage of infarcted area in the tanshinones extracts and phenolic acid extracts groups. Compared with the CMC-Na or saline group: ^###p < 0.001; compared with the AMI group: **p < 0.01, *p < 0.05; the SST groups compared with NST groups: ^△p < 0.05; the tanshinones extracts groups compared with the phenolic acid extracts groups: NS: no significant difference.

Fig. 4

Activities or levels of AST, LDH, SOD, and CK-MB in rats' serum (n = 6) (A–B) Activities of AST in rats' serum in sweated and non-sweated S. miltiorrhiza extracts groups; (C) Comparison of the AST activities between the tanshinones extracts and phenolic acid extracts groups; (D–E) The activities of LDH in rats' serum in sweated and non-sweated S. miltiorrhiza extracts groups; (F) Comparison of the LDH activities between the tanshinones extracts and phenolic acid extracts groups; (G–H) Activities of SOD in rats' serum in sweated and non-sweated S. miltiorrhiza extracts groups; (I) Comparison of the SOD activities between the tanshinones extracts and phenolic acid extracts groups; (J–K) The levels of CK-MB in rats' serum in sweated and non-sweated S. miltiorrhiza extracts groups; (L) Comparison of the SOD activities between the tanshinones extracts and phenolic acid extracts groups. Compared with the CMC-Na or saline group: ^###p < 0.001, ^##p < 0.01; compared with the AMI group: **p < 0.01, *p < 0.05; the SST groups compared with NST groups: ^△p < 0.05; NS: no significant difference.

Fig. 5

The levels of IL-6 and TNF-α in rats' serum (n = 6) (A–B) The levels of IL-6 in rats' serum in sweated and non-sweated S. miltiorrhiza extracts groups; (C) The comparison of the IL-6 levels in the tanshinones extracts and phenolic acid extracts groups; (D–E) The levels of TNF-α in rats' serum in sweated and non-sweated S. miltiorrhiza extracts groups; (F) The comparison of the TNF-α levels in the tanshinones extracts and phenolic acid extracts groups. (G–H) The levels of IL-10 in rats' serum in sweated and non-sweated S. miltiorrhiza extracts groups; (I) The comparison of the IL-10 levels in the tanshinones extracts and phenolic acid extracts groups. Compared with the CMC-Na or saline group: ^###p < 0.001, ^##p < 0.01; compared with the AMI group: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05; the SST-H group compared with NST-H group: ^△p < 0.05; the tanshinones extracts groups compared with the phenolic acid extracts groups: ^&p < 0.05, NS: no significant difference.

Fig. 6

The H&E staining of myocardial tissue of rats in each group (5 × , n = 3).

Fig. 7

The effects of NST and SST on PPARα and RXRα and inflammatory proteins expression (n = 3) (A) Western blotting analysis of the proteins; (B–D) Relative protein densities of p–NF–κB p65/NF-κB p65, RXRα, and PPARα, respectively. The full images of the proteins in (A) were shown in the supplementary data (Figs. S2–S6). Compared with the CMC-Na group: ^#p < 0.05; compared with the AMI group: **p < 0.01, *p < 0.05; the SST-H group compared with NST-H group: ^△p < 0.05; NS: no significant difference.

Fig. 8

The effects of NSPA and SSPA on PPARα and RXRα and inflammatory proteins expression (n = 3) (A) Western blotting analysis of the proteins; (B–D) Relative protein densities of p–NF–κB p65/NF-κB p65, RXRα, and PPARα, respectively; (E–G) The comparison of the protein's levels in the tanshinones extracts and phenolic acid extracts groups. The full images of the proteins in (A) were shown in the supplementary data (Figs. S7–S11). Compared with the saline group: ^#p < 0.05; compared with the AMI group: **p < 0.01, *p < 0.05; the tanshinones extracts groups compared with the phenolic acid extracts groups: ^&&p < 0.01, ^&p < 0.05, NS: no significant difference.

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