Identification of a Sesquiterpene Lactone from Arctium lappa Leaves with Antioxidant Activity in Primary Human Muscle Cells

Abstract

Many pathologies affecting muscles (muscular dystrophies, sarcopenia, cachexia, renal insufficiency, obesity, diabetes type 2, etc.) are now clearly linked to mechanisms involving oxidative stress. In this context, there is a growing interest in exploring plants to find new natural antioxidants to prevent the appearance and the development of these muscle disorders. In this study, we investigated the antioxidant properties of Arctium lappa leaves in a model of primary human muscle cells exposed to H₂O₂ oxidative stress. We identified using bioassay-guided purification, onopordopicrin, a sesquiterpene lactone as the main molecule responsible for the antioxidant activity of A. lappa leaf extract. According to our findings, onopordopicrin inhibited the H₂O₂-mediated loss of muscle cell viability, by limiting the production of free radicals and abolishing DNA cellular damages. Moreover, we showed that onopordopicrin promoted the expression of the nuclear factor-erythroid-2-related factor 2 (Nrf2) downstream target protein heme oxygenase-1 (HO-1) in muscle cells. By using siRNA, we demonstrated that the inhibition of the expression of Nrf2 reduced the protective effect of onopordopicrin, indicating that the activation of the Nrf2/HO-1 signaling pathway mediates the antioxidant effect of onopordopicrin in primary human muscle cells. Therefore, our results suggest that onopordopicrin may be a potential therapeutic molecule to fight against oxidative stress in pathological specific muscle disorders.

Keywords: Arctium lappa L.; antioxidant; burdock leaves; human myoblasts; onopordopicrin.

Figure 1

A. lappa leaves extract protects human myoblasts from induced oxidative stress. Cell death (A) and ROS (B) quantification (percentage of all cells) in human myoblasts that were incubated with the A. lappa leaves extract (BL) at indicated concentrations prior to incubation with H₂O₂. All data represent the means ± SD of three independent experiments; p < 0.001 (***) compared with H₂O₂ (one-way ANOVA).

Figure 2

Bioassay-guided isolation of the antioxidant compound from the A. lappa leaves extract (BL extract). On the left: each step of the BL extract purification; on the right: the associated bioassay on myoblasts with the fractions obtained from the different steps of purification. Cell death quantification (percentage of all cells) in human myoblasts incubated with BL extract fractions at 1 μg/mL (step 1 (A) and 2 (B)) or 0.5 μg/mL (step 3 (C) and 4 (D)) prior to incubation with H₂O₂ All data represent the means ± SD of three independent experiments; p < 0.05 (*) p < 0.01 (**) p < 0.001 (***) compared with H₂O₂ (one-way ANOVA).

Figure 3

Onopordopicrin has a strong antioxidant activity on human muscle cells. Cell death (A,C) and reactive oxygen species (ROS) (B) quantification (percentage of all cells) in human myoblasts treated with onopordopicrin (ONO) at indicated concentrations prior to incubation with lethal concentration of H₂O₂; (D) human myoblasts treated with onopordopicrin (ONO) at indicated concentrations prior to incubation with sub-lethal concentration of H₂O₂. On the left, Western blot analysis of phosphorylated γH2AX protein level; on the right, quantification of the Western blot data, A.U. for arbitrary unit represents the ratio of p-γH2AX protein level on α tubulin protein level. All data represent the means ± SD of three independent experiments; p < 0.01 (**) p < 0.001 (***) compared with H₂O₂ in A, B and C (one-way ANOVA).

Figure 4

Onopordopicrin activates the Nrf2/HO-1 signaling pathway. (A) On the left, Western blot analysis of HO-1 protein level in human myoblast treated with onopordopicrin during 24 h at the indicated concentrations; CTRL: cells not incubated with onopordopicrin; on the right, quantification of Western blot data, A.U. for arbitrary unit represents the ratio of HO-1 protein level on α tubulin protein level. (B) On the left, Western blot analysis of HO-1 and Nrf2 levels in human myoblasts transfected with siNrf2-91 and siNrf2-93; on the right, quantification of the Western blot data, A.U. for arbitrary unit represents the ratio of Nrf2 and HO-1 protein levels on α tubulin protein level. (C,D). Cell death quantification (percentage of all cells) in human myoblasts transfected with Nrf2 siRNA or with siRNA negative control (siCTRL) and treated with dimethylfumarate (DMF) (20 µM) or Tempol (50 µM) or onopordopicrin (0.5 µg/mL) prior to incubation with H₂O₂. All data represent the means ± SD of three independent experiments; p < 0.05 (*) p < 0.001 (***) p < 0.0001 (****) compared to untreated cells in A and compared to siCTRL in B (one-way ANOVA).