Inhibition of TLR4/MAPKs Pathway Contributes to the Protection of Salvianolic Acid A Against Lipotoxicity-Induced Myocardial Damage in Cardiomyocytes and Obese Mice

Abstract

The occurrence of lipotoxicity during obesity-associated cardiomyopathy is detrimental to health. Salvianolic acid A (SAA), a natural polyphenol extract of Salvia miltiorrhiza Bunge (Danshen in China), is known to be cardioprotective. However, its clinical benefits against obesity-associated cardiomyocyte injuries are unclear. This study aimed at evaluating the protective effects of SAA against lipotoxicity-induced myocardial injury and its underlying mechanisms in high fat diet (HFD)-fed mice and in palmitate-treated cardiomyocyte cells (H9c2). Our analysis of aspartate aminotransferase and creatine kinase isoenzyme-MB (CM-KB) levels revealed that SAA significantly reversed HFD-induced myocardium morphological changes and improved myocardial damage. Salvianolic acid A pretreatment ameliorated palmitic acid-induced myocardial cell death and was accompanied by mitochondrial membrane potential and intracellular reactive oxygen species improvement. Analysis of the underlying mechanisms showed that SAA reversed myocardial TLR4 induction in HFD-fed mice and H9c2 cells. Palmitic acid-induced cell death was significantly reversed by CLI-95, a specific TLR4 inhibitor. TLR4 activation by LPS significantly suppressed SAA-mediated lipotoxicity protection. Additionally, SAA inhibited lipotoxicity-mediated expression of TLR4 target genes, including MyD88 and p-JNK/MAPK in HFD-fed mice and H9c2 cells. However, SAA did not exert any effect on palmitic acid-induced SIRT1 suppression and p-AMPK induction. In conclusion, our data shows that SAA protects against lipotoxicity-induced myocardial damage through a TLR4/MAPKs mediated mechanism.

Keywords: MAPKs; TLR4; lipotoxicity; myocardial injury; salvianolic acid A.

FIGURE 1

SAA reverses PA-induced lipotoxicity in H9c2 cardiomyocytes (A) Cardiomyocytes were seeded onto 24-well plates and grown to 80–90% confluence before treatment with the indicated SAA concentrations for 24 h. Cell viability was determined using MTT. (B), (C) Cardiomyocytes were pretreated with indicated SAA concentrations for 1 h and incubated with 0.4 mM palmitic acid (PA) for 12 h and cell viability and LDH release measured. (D) Western blot analysis of cleaved Caspase-3, Bcl-2, and Bax. Values are presented as mean ± SD for three or more independent tests. (E) Hoechst staining for nuclei. Cell death was detected by morphologic examination after Hoechst staining by fluorescence microscopy (mag = 200X). (F) ROS levels were measured using DCFH-DA. (G) MFI analysis of mitochondrial membrane potential using Rh123. Values with different superscripts are significantly different at p < 0.05. * shows comparison with normal control group while # shows comparison with PA-only treatment group.

FIGURE 2

SIRT1/AMPK pathway is not involved in SAA protection from lipotoxicity. Cardiomyocytes were incubated with 0.4 mM palmitic acid (PA) for 12 h. 10 μM SAA was added 1 h before PA treatment. SIRT1, AMPK, and p-AMPK, protein levels were determined by western blot. Data were quantified by densitometric analysis as fold changes. Values are presented as mean ± SD for ≥3 independent tests. Bars with different characters are significantly different, p < 0.05.

FIGURE 3

SAA attenuates palmitate-induced TLR4 activation. Cardiomyocytes were seeded onto 24-well plates and grown to 80% confluence before treatment with 0.4 mM PA for 2, 4, 8 and 12 h (A) or 0.2, 0.4, 0.6, or 0.8 mM PA for 4 h (B) For the detection of anti-lipotoxicity role of SAA, H9c2 cells were incubated with 0.4 mM palmitic acid (PA) for 4 h. 10 μM SAA was added 1 h before free fatty acids treatment. Representative western blot data on TLR4 levels. Data are shown as mean ± SD. Total protein was extracted from myocardial cells. TLR4, MyD88, p-JNK, and p-ERK1/2 levels were determined by western blot. Values with different superscripts are significantly different at p < 0.05. * indicates comparison with normal control group while # indicates comparison with PA-only treatment group.

FIGURE 4

TLR4 was involved in lipotoxicity-induced injury in cardiomyocytes. Cardiomyocyte cells were treated with PA at 0.4 mM for 4 h with or without 1 h CLI-095 preincubation (1 μM). PA-induced cytotoxicity in CLI-095-treated cardiomyocytes was determined using the MTT assay (A) and analysis of LDH release into culture medium (B). (C) Nuclear staining with Hoechst. (D) Representative western blot with densitometric analysis of cleaved Caspase-3 levels in cells. (E) ROS levels were measured using DCFH-DA. (F) MFI analysis of mitochondrial membrane potential using Rh123. Values are presented as mean ± SD for ≥3 independent tests. * indicates comparison with normal controls while # shows comparison with PA-only treatment. Bars with different characters are significantly different, p < 0.05.

FIGURE 5

TLR4 activation contributes to SAA protection from lipotoxicity-induced injury in cardiomyocytes. Cardiomyocyte cells were treated with PA at 0.4 mM for 4 h. TLR4 was activated with 100 ng/ml LPS for 2 h before PA treatment. 10 μM SAA was added 1 h before PA treatment. (A) Cell viability was tested using MTT. (B) LDH release measurement. (C) Nuclear staining with Hoechst. Cell death was detected by assessing nuclear morphology by fluorescence microscopy at a ×200 magnification (D) ROS levels were measured by DCFH-DA. (E) Mitochondrial membrane potential (MMP) was examined by fluorescence microscopy. (F) Cleaved Caspase-3, TLR4, MyD88, p-JNK, and p-ERK1/2 levels were determined by western blot. Values are presented as mean ± SD for ≥3 independent tests. Bars with different characters are significantly different, p < 0.05.

FIGURE 6

TLR4/MAPKs pathway is involved in SAA protection of cardiomyocytes from lipotoxicity-induced injury in high-fat diet (HFD)-fed mice. Animals were divided into the following groups (6 mice each); normal diet group (Control), HFD group (HFD), normal diet with 40 mg/kg BW SAA group (SAA), and HFD with 40 mg/kg BW SAA (SAA + HFD) group. The Control group was fed AIN-93G diet. HFD mice were fed 60% fat diet to induce obesity (60% fat, D12492, Research Diets, New Brunswick, NJ) and water enriched with high fructose and sucrose. SAA was dissolved in sterilized physiologic saline with a stock concentration of 20 mg/mL. A total volume of 100 μL SAA diluted solution or sterilized physiologic saline was intraperitoneally administered every other day for 12 weeks. (A) H/E staining of cardiomyocytes - scale bars = 50 μm. (B) Plasma aspartate aminotransferase (AST) level (C) plasma creatine kinase, MB (CK-MB) level. (D) Cleaved Caspase-3, TLR4, MyD88, JNK, p-JNK, ERK1/2 and p-ERK1/2 levels in mouse heart tissues were determined by western blot. Values with different superscripts are significantly different at p < 0.05. * indicates comparison with normal controls while # shows comparison with HFD-fed group.