The endophytic fungus Penicillium oxalicum isolated from Ligusticum chuanxiong Hort possesses DNA damage-protecting potential and increases stress resistance properties in Caenorhabditis elegans

Abstract

The chemical composition and antioxidant activity of extracts (POE) of Penicillium oxalate isolated from Ligusticum chuanxiong Hort have been investigated. However, the biological activity of POE is limited, and its antioxidant, stress resistance and DNA protection effects in vivo are unclear. The current study aims to explore the beneficial effects of POE on DNA damage protection in pBR322 plasmid and lymphocytes and stress resistance in Caenorhabditis elegans. The results showed that POE increased the survival rate of C. elegans under 35°C, UV and H₂O₂ stress, attenuated ROS and MDA accumulation, and enhanced the activity of some important enzymes (SOD, CTA, and GSH-PX). In addition, the POE-mediated stress resistance involved the upregulation of the expression of the sod-3, sod-5, gst-4, ctl-1, ctl-2, daf-16, hsp-16.1, hsp-16.2, and hsf-1 genes and acted dependently on daf-16 and hsf-1 rather than skn-1. Moreover, POE also reduced lipofuscin levels, but did not prolong the lifespan or damage the growth, reproduction and locomotion of C. elegans. Furthermore, POE showed a protective effect against DNA scission in the pBR322 plasmid and lymphocytes. These results suggested that P. oxalate extracts have significant anti-stress and DNA protection potential and could be potential drug candidates in the pharmaceutical field, thus greatly broadening the understanding of the biological effects of the endophytic fungus P. oxalate.

Keywords: Caenorhabditis elegans; DNA damage protection; Penicillium oxalicum; antioxidant; endophytic fungi; oxidative stress resistant.

FIGURE 1

The effect of POE on stress resistance in C. elegans. (A) Survival curve of worms under UV irradiation-induced stress. (B) Survival curve of worms under H₂O₂-induced stress. (C) Survival curve of worms under 35°C-induced stress. Three independent biological replicates were performed. Differences compared to control group were considered significant at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).

FIGURE 2

The effect of POE on the antioxidant defense system in C. elegans under normal and H₂O₂-induced oxidative stress conditions. (A) The MDA content. (B) The SOD activity. (C) The GSH-Px activity. (D) The CAT activity. Bars with no letters in common are significantly different (p < 0.05).

FIGURE 3

The effect of POE on intracellular levels of ROS in C. elegans. (A) Accumulation of ROS in C. elegans under normal conditions. (B) Accumulation of ROS in C. elegans under H₂O₂-induced oxidative stress. (i): Relative fluorescence intensity of worms was quantified using ImageJ software. (ii): Representative image of worms which was treated with POE in both conditions. Bars with no letters in common are significantly different (p < 0.05).

FIGURE 4

The effect of POE on lipofuscin accumulation and body size in C. elegans. (A) Representative images of fluorescence and bright field micrographs are shown, the scale bar was 100 μm; (B) body length and lipofuscin was measured and quantitated by ImageJ. Bars with no letters in common are significantly different (p < 0.05).

FIGURE 5

The effect of POE on lifespan of C. elegans. Results of lifespan experiments were analysed using the Kaplan-Meier survival model, and for significance by means of a long rank pairwise comparison test between the control and treatment groups. Differences compared to control group were considered significant at p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***).

FIGURE 6

The effect of POE on reproduction and movement in C. elegans. (A) Brood size; (B) Progeny number; (C) Hatchability; (D) The three levels of locomotivity were measured and the individuals were classified according to the movement: A-free movement, B-movement after prodding, C-weak movement after prodding. Data were expressed as the mean ± SD (n = 3). Bars with different letters indicated statistical significance (p < 0.05).

FIGURE 7

The molecular mechanism of POE in the antioxidant stress. (A) The expression of stress-related genes in C. elegans under H₂O₂-induced oxidative stress conditions. (B) The survival curve of skn-1 mutant worms under H₂O₂-induced oxidative stress. (C) The survival curve of hsf-1 mutant worms under H₂O₂-induced oxidative stress. (D) The survival curve of daf-16 mutant worms under H₂O₂-induced oxidative stress. Data were expressed as the mean ± SD (n = 3). Bars with different letters indicated statistical significance (p < 0.05). * Significant p-value <0.05 by the log-rank test.

FIGURE 8

The DNA protective effect of POE against •OH generated by Fenton’s reagent. (A) Electrophoretogram. Lanes 1 and 2 were the normal DNA treated with and without 1 mM FeSO₄ and 1 mM H₂O₂, respectively. Lanes 3–6 were treated with various concentrations of POE (25, 50, and 75 μg/ml) and VE (500 μM). (B) Double helix percentage. Bars with different letters indicated statistical significance (p < 0.05).

FIGURE 9

The effect of POE on DNA damage protection in lymphocyte. (A) Various forms of DNA damage seen as (a) MN, (b) nucleoplasmic ridge and (c) nuclear bud on cells treated with H₂O₂, the scale bar was 5 μm. (B) The mean frequency of DNA damage in human lymphocytes exposed to H₂O₂ (250 µM), H₂O₂ (250 µM) + POE (25, 50, and 75 μg/ml). Data were expressed as the mean ± SD (n = 3). Bars with different letters indicated statistical significance (p < 0.05).

FIGURE 10

A possible model of the mechanism of action of POE-mediated stress resistance in C. elegans. POE alleviates the accumulation of ROS by activating the antioxidant defense system, and ultimately improves the anti-stress ability of C. elegans. The observed effects were mediated, at least in part, by the two master regulators DAF-16 and HSF-1 signaling pathways rather than SKN-1.