International Journal of
Molecular Sciences Article
A Polysaccharide Purified from Morchella conica Pers. Prevents Oxidative Stress Induced by H2O2 in Human Embryonic Kidney (HEK) 293T Cells Na Xu 1 , Yi Lu 2 , Jumin Hou 3 , Chao Liu 4 and Yonghai Sun 1, * 1 2 3 4
*
College of Food Science and Engineering, Jilin University, Changchun 130062, China;
[email protected] Jilin Provincial Key Laboratory of Animal Embryo Engineering, Jilin University, Changchun 130062, China;
[email protected] College of Food Science and Engineering, Changchun University, Changchun 130028, China;
[email protected] School of Food Engineering, Jilin Agriculture Science and Technology College, Jilin 132101, China;
[email protected] Correspondence:
[email protected]; Tel.: +86-431-8509-4968
Received: 22 November 2018; Accepted: 11 December 2018; Published: 13 December 2018
Abstract: Morchella conica Pers. (M. conica) has been used both as a medical and edible mushroom and possesses antimicrobial properties and antioxidant activities. However, the antioxidant properties of polysaccharides purified from M. conica have not been studied. The aim of this study was to investigate the in vitro antioxidant properties of a polysaccharide NMCP-2 (neutral M. conica polysaccharides-2) purified from M. conica, as determined by radical scavenging assay and H2 O2 -induced oxidative stress in HEK 293T cells. Results showed that NMCP-2 with an average molecular weight of 48.3 kDa possessed a much stronger chelating ability on ferrous ions and a higher ability to scavenge radical scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH) than the other purified fraction of NMCP-1 from M. conica. Moreover, 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetra-zolium bromide (MTT) assay showed that NMCP-2 dose-dependently preserved cell viability of H2 O2 -induced cells. The NMCP-2 pretreated group reduced the generation of reactive oxygen species (ROS) content and increased the mitochondria membrane potential (MMP) levels. In addition, Hoechst 33342 staining revealed cells treated with NMCP-2 declined nuclear condensation. Ultrastructural observation revealed that NMCP-2 pretreatment alleviated the ruptured mitochondria when exposed to H2 O2 . Furthermore, western blot analysis showed that NMCP-2 prevented significant downregulation of the protein expression of Bax, cleaved caspases 3, and upregulated Bcl-2 levels. These results suggest the protective effects of NMCP-2 against H2 O2 -induced injury in HEK 293T cells. NMCP-2 could be used as a natural antioxidant of functional foods and natural drugs. Keywords: Morchella conica; polysaccharides; oxidative stress
1. Introduction Oxidative stress has been implicated in several chronic diseases that include aging, cancer, diabetes, cardiovascular diseases, and neurodegenerative diseases [1]. Oxidative stress is a condition of imbalance between prooxidants and antioxidants, which is mainly caused by the excessive accumulation of reactive oxygen species (ROS), such as hydrogen peroxide (H2 O2 ), hydroxyl free radicals (• OH), and hydroxyl free radicals (O2 − ) [2]. The excessive ROS can pass through the cell membrane and cause oxidative damage to lipids, proteins, and DNA, thereby inducing cell apoptosis
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through different metabolic pathways [1,3]. Thus, the scavenging of ROS mediated by antioxidants mightthrough be a promising treatmentpathways option for retarding oxidative stress-related diseases [4]. different metabolic [1,3]. Thus, the scavenging of ROS mediated by antioxidants Edible fungi or mushrooms rich for in nutrition, and possess notable diseases medicinal might be a promising treatmentare option retarding oxidative stress-related [4].properties and Edible or mushrooms rich in nutrition, and possessinnotable and bioactivities [5]. fungi The most abundantarebiological macromolecules ediblemedicinal fungi areproperties polysaccharides. [5]. The mostof abundant biological macromolecules fungi are They bioactivities have a wide range biological activities, includingin edible antitumor [6],polysaccharides. antimicrobial [7], They have a wide range of [9], biological activities, activities including [10]. antitumor [6], conica antimicrobial immunomodulatory [8], prebiotic and antioxidant Morchella Pers. (M.[7], conica) immunomodulatory [8], prebiotic [9], and antioxidant activities [10]. Morchella conica Pers. (M. conica) is a rare edible mushroom belonging to the genus Morchella, well known for its delicate taste and is a rare edible mushroom belonging to the genus Morchella, well known for its delicate taste and unique appearance [4,11]. Previous studies have showed the M. conica extracts play an important unique appearance [4,11]. Previous studies have showed the M. conica extracts play an important role role in scavenging free radicals [12]. However, reports about the antioxidant activities of purified in scavenging free radicals [12]. However, reports about the antioxidant activities of purified polysaccharides fromfrom M. conica and the effectson onpreventing preventing oxidative stress polysaccharides M. conica and theevaluation evaluation of of their their effects oxidative stress are are barelybarely mentioned. mentioned. In theInpresent study, we we analyzed the andpreliminary preliminary structural features the present study, analyzed thechemical chemical composition composition and structural features of purified (M. conica polysaccharides) fractionofofneutral neutralM. M. conica conica polysaccharides-2 (NMCPof purified MCPMCP (M. conica polysaccharides) fraction polysaccharides-2 (NMCP-2). 2). We investigated the protective effect of NMCP-2 2O2-induced oxidative stressininHEK HEK293T 293T cells We investigated the protective effect of NMCP-2 on Hon oxidative stress 2 OH 2 -induced cells and analyzed its effects on cell viability, the generation of ROS, apoptosis, and the mechanisms and analyzed its effects on cell viability, the generation of ROS, apoptosis, and the mechanisms in vitro. in vitro.
2. Results and Discussions
2. Results and Discussions
2.1. Purification of Crude MCP
2.1. Purification of Crude MCP
The crude MCP was separated through a DEAE-52 cellulose column, fractionated into two The crude MCP was separated through a DEAE-52 cellulose column, fractionated into two polysaccharide peaks designated as NMCP, AMCP (acidic M. conica polysaccharides) (Figure 1a). polysaccharide peaks designated as NMCP, AMCP (acidic M. conica polysaccharides) (Figure 1a). The main fraction (NMCP) was collected withSephadex Sephadex G-100 filtration The main fraction (NMCP) was collectedand andfurther further purified purified with G-100 gel gel filtration chromatography, affording two independent elutionpeaks peaks NMCP-1 NMCP-2 chromatography, affording two independent elution of of NMCP-1 and and NMCP-2 (Figure(Figure 1b). In 1b). In thisthis study, NMCP-1 werecollected collectedforfor further radical scavenging analysis. study, NMCP-1and andNMCP-2 NMCP-2 were further radical scavenging analysis.
Figure 1. The elution curve of polysaccharides isolated from the M. conica on a DEAE-52 cellulose Figure 1. The elution curve of polysaccharides isolated from the M. conica on a DEAE-52 cellulose column. (a) The cellulose column 0–0.25mol/L mol/L linear gradient of NaCl column. (a) DEAE-52 The DEAE-52 cellulose columnwas waseluted eluted with with aa 0–0.25 linear gradient of NaCl at at a flowa rate of 1 mL/min. The polysaccharide fractions were pooled and named as neutral M. conica flow rate of 1 mL/min. The polysaccharide fractions were pooled and named as neutral M. conica polysaccharides (NMCP) and and AMCP, respectively. (b)(b) Elution SephadexGG-100 polysaccharides (NMCP) AMCP, respectively. Elutioncurve curveofofthe theNMCP NMCP on a Sephadex column. Sephadex G-100G-100 column waswas eluted with distilled ataaflow flowrate rate mL/min. 100 The column. The Sephadex column eluted with distilledwater water at of of 0.3 0.3 mL/min. Thepolysaccharide two polysaccharide fractions were namedNMCP-1 NMCP-1 and and NMCP-2, The two fractions were named NMCP-2,respectively. respectively.
2.2. DPPH (2,2-diphenyl-1-picrylhydrazyl) Scavenging and Ferrous FerrousIon IonChelating Chelating Ability of NMCP-1 2.2. DPPH (2,2-diphenyl-1-picrylhydrazyl) Scavenging Effect Effect and Ability of NMCP-1 and NMCP-2 and NMCP-2 DPPH is a stable radical that hasbeen beenextensively extensively used radical elimination reactions. DPPH is a stable freefree radical that has usedfor forfree free radical elimination reactions. Free radicals scavengedwhen whenthey they encounter electron or hydrogen donordonor [13]. It [13]. can beItseen Free radicals are are scavenged encounteranan electron or hydrogen can be from Figure 2a that the DPPH radical scavenging abilities of NMCP-1 and NMCP-2 were doseseen from Figure 2a that the DPPH radical scavenging abilities of NMCP-1 and NMCP-2 were dependent when comparison with the same concentrations of Vitamin c (Vc). At the concentration of dose-dependent when comparison with the same concentrations of Vitamin c (Vc). At the concentration of 4 mg/mL, the scavenging activities of NMCP-1 and NMCP-2 are 48.29 ± 4.61% and 73.49 ± 6.14%, respectively. The DPPH scavenging ability in NMCP-2 at six concentrations from 0.1 to 4 mg/mL was
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4 mg/mL, the scavenging activities of NMCP-1 and NMCP-2 are 48.29 ± 4.61% and 73.49 ± 6.14%, respectively. The DPPH ability in NMCP-2 at six concentrations from(p 0.1
