Phenolic Acids-Rich Fractions from Agaricus bitorguis (Quél.) Sacc. Chaidam ZJU-CDMA-12 Mycelia Modulate Hypoxic Stress on Hypoxia-Damaged PC12 Cells

Abstract

Hypoxia is a common pathological process in various clinical diseases. However, there is still a lack of effective anti-hypoxia active substances. Agaricus bitorguis (Quél.) Sacc Chaidam (ABSC) is a rare wild edible macrofungus that grows underground at high altitudes. Herein, intracellular phenolic acids-rich fractions (IPA) were extracted from ABSC ZJU-CDMA-12, and the structural characterization and anti-hypoxia activity of IPA on PC12 cells were elucidated as well. The results of HPLC-Q-TOF-MS illustrated that five kinds of IPA were isolated from ABSC, including (-)-epicatechin gallate, arabelline, yunnaneic acid D, 2'-O-p-hydroxybenzoyl-6'-O-trans-caffeoylgardoside,4'-O-methylgallocatechin-(4->8)-4'-O-methylepigallocatechin. IPA extracted from ABSC proved to show anti-hypoxia activity on hypoxia-damaged PC12 cells. Hypoxia enhanced reactive oxygen species (ROS) generation and reduced the mitochondrial membrane potential (ΔΨm) in PC12 cells, resulting in the inhibition of survival and induction of apoptosis in PC12 cells. Measurements of 100 μg/mL and 250 μg/mL IPA could significantly reduce hypoxia-induced damage in PC12 cells by decreasing overproduced intracellular ROS, improving ΔΨm, and reducing cell apoptosis rate. Our findings indicated that the IPA from ABSC potentially could be used as novel bioactive components applied to anti-hypoxia functional foods or medicines.

Keywords: Agaricus bitorquis (QuéL.) Sacc. Chaidam; anti-hypoxia activity; intracellular phenolic acids; structure characterization.

Figure 1

The chromatogram of IPA isolated from ABSC.

Figure 2

Hypoxia induced PC12 cell injury and IPA promote the survival of hypoxia-damaged PC12 cells. (A) Effect of oxygen contents and treatment time on the survival of PC12 cells; (B) Protective effect of IPA on hypoxia-damaged PC12 cells, as determined by CCK-8 assay. * p < 0.05, ** p < 0.01 indicates a significant difference versus the control group.

Figure 3

Fluorescence micrographs of PC12 cells stained with Hoechst 33342 and SYTOX green staining (original magnification, ×100).

Figure 4

IPA protects hypoxia-damaged PC12 cells from overproduced ROS and mitochondrial damage. (A) Representative images showing the effects of IPA on reducing the overproduced ROS by DCFH-DA staining and the mitochondrial membrane potential (ΔΨm) in hypoxia-damaged PC12 cells as determined by Rhodamine-123 (original magnification, × 100). (B) The ROS production for each PC12 cell treatment. (C) The ΔΨm for each PC12 cell treatment. Data are expressed as the mean ± SD (n = 3; one-way analysis of variance followed by the T-test). The different uppercase letters A, B, C indicate significant differences, all experiments were conducted in triplicate. ** p < 0.01 indicates a significant difference versus the control group. ## p < 0.01 indicates a significant difference versus the 0 group under hypoxia condition.

Figure 5

IPA reduce hypoxia-induced apoptosis rate of PC12 cells. (A) Cell apoptosis rate was determined by flow cytometry. (B) The apoptosis rate for each PC12 cell treatment. Data are expressed as the mean ± SD (n = 3; one-way analysis of variance followed by the T-test). All experiments were conducted in triplicate. * p < 0.05, ** p < 0.01 indicates a significant difference versus the control group. # p < 0.05, ## p < 0.01 indicates a significant difference versus the 0 group under hypoxia condition.