Beta carotene protects H9c2 cardiomyocytes from advanced glycation end product-induced endoplasmic reticulum stress, apoptosis, and autophagy via the PI3K/Akt/mTOR signaling pathway

Abstract

Background: Diabetic cardiomyopathy (DCM), which is associated with many pathological processes, commonly occurs when advanced glycation end products (AGEs) are present. β-carotene (BC) is a well-known vitamin A precursor that is found in many fruits and vegetables. BC can reduce the risk of cancer and cardiovascular disease. This study aimed to investigate the effect of BC on AGE-induced myocardial injury in vitro.

Methods: Cell viability test was used to select 40 µM concentrations of BC to treat AGE-induced H2c9 cells. The cell apoptosis was detected by flow cytometry. Western blotting was used to measure the protein expression levels of Bcl-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), cleaved caspase-3, activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), beclin 1, p62,microtubule-associated protein 1 light chain 3 (LC3), phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-AKT), and phosphorylated mTOR (p-mTOR). Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of lactate dehydrogenase (LDH) and cardiac troponin-1 (cTn-I). Reactive oxygen species (ROS) was detected by flow cytometry. The levels of malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) were used to determine MDA kits, SOD assay kit and GSH-Px kit, respectively.

Results: BC significantly inhibited AGE-induced cell death and apoptosis in H9c2 cells. BC had a suppressive effect on intracellular ROS production and antioxidative enzyme reduction. Moreover, BC decreased hyperactive endoplasmic reticulum (ER) stress and autophagy in H9c2 cells. Furthermore, BC exerted a cardioprotective effect in AGE-induced H9c2 cells via the activation of the PI3K/Akt/mTOR signaling pathway.

Conclusions: BC exhibited a cardioprotective effect AGE-induced apoptosis. Our study provides a foundation for further study into the potential value of BC for treating DCM or other heart diseases.

Keywords: Apoptosis; autophagy; beta carotene; endoplasmic reticulum stress (ER stress); phosphatidyl inositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR).

Figure 1

BC protects against AGE-induced cell death. (A) H9c2 cells were treated with series doses of BC for 24 h. Cell viability was measured by MTT assay. (B) H9c2 cells were pre-treated with different concentrations of BC, then stimulated with AGEs (200 µg/mL) for 24 h. Cell viability was measured by MTT assay. (C,D) The effect of BC on LDH (C) and cTn-I (D) leakage in AGE-induced H9c2 cells. Each experiment was performed in triplicate. *, P<0.05 vs. control group; ^#, P<0.05; ^##, P<0.01 vs. AGE-induced group. BC, beta-carotene; AGEs, advanced glycation end products; LDH, lactate dehydrogenase; cTn-I, cardiac troponin-1.

Figure 2

BC attenuates AGE-induced cell apoptosis. (B) H9c2 cells were pre-treated with BC (40 µM) and then stimulated with AGEs (200 µg/mL) for 24 h. (A,B) Cell apoptosis was detected by Annexin V flow cytometry. (C) The protein expression levels of Bax, Bcl-2, and cleaved caspase-3 were detected by Western blot. (D,E) Semi-quantitative analysis of the relative levels of Bax, Bcl-2, and cleaved caspase-3. Each experiment was performed in triplicate. β-actin was used as loading control. *, P<0.05 vs. control group; ^#, P<0.05 vs. AGE-induced group. BC, beta-carotene; AGEs, advanced glycation end products; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma-2.

Figure 3

BC suppresses AGE-induced cell oxidative stress. H9c2 cells were pre-treated with BC (40 µM) and then stimulated with AGEs (200 µg/mL) for 24 h. (A) Intracellular ROS was measured with DCFH-DA. The data were obtained by flow cytometry. (B) The production of MDA. (C,D) The activity of GSH-Px (C) and SOD (D). Each experiment was performed in triplicate. *, P<0.05 vs. control group; ^#, P<0.05 vs. AGE-induced. BC, beta-carotene; AGEs, advanced glycation end products; ROS, Reactive oxygen species; DCFH-DA, dichloro-dihydro-fluorescein diacetate; MDA, malondialdehyde; GSH-Px, glutathione peroxidase; SOD, superoxide dismutase.

Figure 4

BC alleviates AGE-induced elevation of ER stress. H9c2 cells were pre-treated with BC (40 µM) and then stimulated with AGEs (200 µg/mL) for 24 h. (A) The protein level of ATF4, GRP78, and CHOP was detected by Western blot. (B,C,D) Quantification of Figure 4A. Each experiment was performed in triplicate. *, P<0.05 vs. control group; ^#, P<0.05 vs. AGE-induced group. BC, beta-carotene; AGEs, advanced glycation end products; ER, endoplasmic reticulum; ATF4, activating transcription factor 4; GRP78, glucose-regulated protein 78; CHOP, CCAAT/enhancer-binding protein homologous protein.

Figure 5

BC inhibits AGE-induced autophagy. H9c2 cells were pre-treated with BC (40 µM) and then stimulated with AGEs (200 µg/mL) for 24 h. (A) The protein levels of beclin 1, p62, and LC3 were detected by Western blot. (B,C,D) Semi-quantitative analysis of the relative level of beclin 1, p62, and LC3. (E) Representative images of intracellular LC3 redistribution (green spots) in H9c2 cells under different conditions. (F) The quantification of the number of LC3 puncta. Each experiment was performed in triplicate. *, P<0.05 vs. control group; ^#, P<0.05 vs. AGE-induced group. BC, beta-carotene; AGEs, advanced glycation end products; LC3, microtubule-associated protein 1 light chain 3.

Figure 6

BC activates AGE-induced inhibition of the PI3K/Akt/mTOR signaling pathway. H9c2 cells were pre-treated with BC (40 µM) and then stimulated with AGEs (200 µg/mL) for 24 h. (A) The expressions of p-PI3K, p-AKT and p-mTOR were detected by Western blot. (B,C,D) Semi-quantitative analysis of the relative levels ofp-PI3K, p-AKT, and p-mTOR. Each experiment was repeated in triplicate. *, P<0.05 vs. control group; ^#, P<0.05 vs. AGE-induced group. BC, beta-carotene; AGEs, advanced glycation end products; PI3K/Akt/mTOR, Phosphatidyl inositol 3-kinase/Akt/ mammalian target of rapamycin; p-PI3K, phosphorylated PI3K; p-AKT, phosphorylated Akt; p-mTOR, phosphorylated mTOR.

Figure 7

PI3K/Akt/mTOR is essential in BC protection of AGE-induced cardiac injuries. H9c2 cells were pre-treated with BC (40 µM) and LY294002 (LY, 10 µM) and then stimulated with AGEs (200 µg/mL) for 24 h. (A) The expression levels of Bax and Bcl-2 were detected by Western blot. (B) Semi-quantitative analysis of the relative levels of Bax and Bcl-2. (C) Cell apoptosis rate. (D) Intracellular ROS production. (E) SOD activity. (F) The expression levels of ATF4, GRP78, and CHOP were detected by Western blot. (G) Semi-quantitative analysis of the relative levels ofATF4, GRP78, and CHOP. (H) The expression levels of beclin 1, p62, and LC3 were detected by Western blot. (I) Semi-quantitative analysis of the relative level of beclin 1, p62 and LC3. (J) The quantification of the number of LC3 puncta. Each experiment was performed in triplicate. *, P<0.05 vs. control; ^#, P<0.05 vs. AGEs; ^$, P<0.05 vs. AGEs + BC. PI3K/Akt/mTOR, Phosphatidyl inositol 3-kinase/Akt/mammalian target of rapamycin; BC, beta-carotene; AGEs, advanced glycation end products; Bax, Bcl-2-associated X protein; Bcl-2, B-cell lymphoma-2; ROS, reactive oxygen species; SOD, superoxide dismutase; ATF4, activating transcription factor 4; GRP78, glucose-regulated protein 78; CHOP, CCAAT/enhancer-binding protein homologous protein; LC3, microtubule-associated protein 1 light chain 3.