Functional Mechanisms of Dietary Crocin Protection in Cardiovascular Models under Oxidative Stress

Abstract

It was previously reported that crocin, a water-soluble carotenoid isolated from the Crocus sativus L. (saffron), has protective effects on cardiac cells and may neutralize and even prevent the formation of excess number of free radicals; however, functional mechanisms of crocin activity have been poorly understood. In the present research, we aimed to study the functional mechanism of crocin in the heart exposed to oxidative stress. Accordingly, oxidative stress was modeled in vitro on human umbilical vein endothelial cells (HUVECs) and in vivo in mice using cellular stressors. The beneficial effects of crocin were investigated at cellular and molecular levels in HUVECs and mice hearts. Results indicated that oral administration of crocin could have protective effects on HUVECs. In addition, it protects cardiac cells and significantly inhibits inflammation via modulating molecular signaling pathways TLR4/PTEN/AKT/mTOR/NF-κB and microRNA (miR-21). Here we show that crocin not only acts as a direct free radical scavenger but also modifies the gene expression profiles of HUVECs and protects mice hearts with anti-inflammatory action under oxidative stress.

Keywords: cardiovascular; crocin; dietary antioxidants; microRNA; oxidative stress.

Figure 1

Antioxidant activity of crocin. (A) The antioxidant capacity of crocin was evaluated by DPPH. The IC₅₀ (concentration required to inhibit 50% of DPPH radicals) of crocin was ~1000 µM. (B) Kinetics of fluorescence decay measured by the oxygen radical antioxidant capacity (ORAC) assay. (C) HUVEC cell viability determination by MTT assay at 24, 48, and 72 h after treatment with 125, 250, 500, 1000, 2000, and 3000 µM of crocin. (D,E) Effects of different concentrations of AAPH (D) and H₂O₂ (E) on cell viability. (F,G) Effects of different concentrations of crocin and AAPH 5 µM (F,G) H₂O₂ 200 µM on cell viability. Crocin 500 μM protects cells against AAPH and H₂O₂ toxic effects and increases HUVEC viability. The data show the mean values ± SD of at least three independent experiments. (D,E) * represents statistical significance p < 0.05, ** p < 0.01, *** p < 0.001 compared to control (Cnt). (F,G) Levels not connected by the same symbol (+, *, **, ***) are significantly different (p < 0.05).

Figure 2

Protective effect of crocin on HUVEC DNA damage. (A–F) DAPI staining (magnification 40×) and (a–f) light microscope (magnification 20×). (A,a) control cells; (B,b) crocin-treated cells; (C,c) AAPH+crocin-treated cells; (D,d) H₂O₂+crocin-treated cells; (E,e) AAPH-treated cells; and (F,f) H₂O₂-treated cells.

Figure 3

Antioxidant effects of crocin on HUVEC cells. (A) Crocin protects HUVECs DNA against AAPH and H₂O₂, as evaluated by the DNA ladder assay. (B,C) Crocin exerts antioxidant properties through modulating Nrf2, HO1, and NQO1 (B) mRNA and (C) protein levels. Effects of crocin on HUVEC cell survival and apoptosis. (D,F) mRNA and (E,G) protein levels of AKT, mTOR, PTEN, BAX, and BCL-XL in HUVEC-treated cells. The 2^−ΔΔCt method was used to calculate the relative expression (fold change) between sample groups. Relative expression is indicated as the mean (SD) of the log (₂) fold change. GAPDH was used as an endogenous control. Cr: crocin. NS: not significant. The data show the mean values ± SD of at least three independent experiments. * p < 0.01, ** p < 0.001.

Figure 4

The effect of crocin on different phases of the cell cycle under oxidative stress. HUVECs were treated with IC₅₀ concentrations of AAPH and H₂O₂ for 48 h, stained with PI, and analyzed by flow cytometry. The figure shows the quantitative distribution of HUVECs in different phases of the cell cycle in the untreated cells, the cells treated with AAPH and H₂O₂, and the cells co-treated with crocin and stressors. Red circles indicate apoptotic cells in sub G1 population. Crocin treatment could decrease the number of apoptotic cells in sub G1 population compared to AAPH and H₂O₂ groups in flow cytometric analysis.

Figure 5

Rhodamine 123 staining for mitochondrial membrane potential. The figure represents the untreated HUVECs, which show high fluorescence, indicating a polarized mitochondrial membrane. AAPH- and H₂O₂-treated cells show low fluorescence (magnification 20×). It means that oxidative stress could affect ATP synthesis, and crocin co-treated modified the mitochondrial membrane potential. Cr: Crocin.

Figure 6

Crocin’s protective effects on stressed-out mice. (A) Experimental setting: four groups of mice receiving diet by gavage. GI and GII received a normal feed, and in GIII and GIV, crocin (50 mg/kg). After 14 days, the GII and GIII were injected intraperitoneally with AAPH (5 mg/kg), and the samples were collected after 6 h of stress treatment. (B) Immunohistological Nrf2 staining in three different zones of the mouse heart under AAPH-induced oxidative stress: non-treated (GII) or treated with crocin (GIV) and control (GI); the yellow arrow indicates nuclear positive staining; the positive basal labeling is attributed to muscle fiber positive staining concentrated within the intercalated disc. (C) Serum biochemical parameters TAS: Total Antioxidant Status, Trig: triglyceride; HDL: high-density lipoprotein, Chol: cholesterol, MDA: Malondialdehyde. The data show mean values ± SD of at least three independent measurements of serum parameters.

Figure 7

The signaling pathway is affected by crocin in mice hearts (A) mRNA levels of TLR4, MYD88, and NF-κB, (B) MYD88 protein, and (C) miR-21 transcriptional levels in stress-induced mice hearts. The 2^-ΔΔCt method was used to calculate the relative expression (fold change) between sample groups. GAPDH and U6 were used as internal controls, respectively, for mRNA and protein, as well as miR-21 normalization. Fold change is indicated as the mean (SD) of log (2). The data show mean values ± SD of at least three independent experiments. * p < 0.01, ** p < 0.001. NS: not significant.

Figure 8

Suggested schematic illustration of the mechanism by which crocin could induce cell survival and exert anti-oxidative and anti-inflammatory effects in stressed cardiomyocytes. In response to the treatment of AAPH and H₂O₂, crocin represents antioxidant and anti-inflammatory properties through activation of Nrf2 and inhibition of NF-κB, respectively. Activation of Nrf2 is required for the cellular antioxidant activity of crocin. In response to the AAPH-induced stress, crocin acts as a free radical scavenger, and it somehow modulates Nrf2/HO1/NQO1 expression. Inhibition of NF-κB, either through TLR4 and MYD88 or other related signaling, decreases transcription of pro-inflammatory genes and miR-21 and overall declines inflammation. Moreover, crocin hinders PTEN from surviving cells against oxidative stress. A decreased level of PTEN significantly induces AKT expression at both transcriptional and translational levels, which stimulates mTOR signaling and leads to cell proliferation and survival. Moreover, crocin induces anti-apoptotic factors to block mitochondrial-induced cell apoptosis. Since AAPH induces ROS in the cell membrane and cytoplasm but not directly in mitochondria, the molecular signaling pathway induced by AAPH may be different from H₂O₂; therefore, it is rational to observe differences in gene expression levels in response to crocin treatment in AAPH and H₂O₂-induced stressed-out cardiomyocytes (see text). mTOR: protein kinase mammalian target of rapamycin, TLR4: tool-like receptor 4, Nrf-2: nuclear factor-erythroid 2-related factor 2, HO-1: heme oxygenase-1, NQO1: NAD quinone oxidoreductase 1, ROS: reactive oxygen species.