Emodin induces human T cell apoptosis in vitro by ROS-mediated endoplasmic reticulum stress and mitochondrial dysfunction

Abstract

Aim: To elucidate the molecular mechanisms underlying the immunosuppressive effects of emodin isolated from Rheum palmatum L.

Methods: Human T cells were isolated from the peripheral venous blood of 10 healthy adult donors. Cell viability was analyzed with MTT assay. AO/EB and Annexin V/PI staining and DNA damage assay were used to detect cell apoptosis. Fluorescence staining was used to detect the levels of ROS, the mitochondrial membrane potential and intracellular Ca(2+). Colorimetry was used to detect the levels of MDA and total SOD and GSH/GSSG ratio. The expression and activity of caspase-3, -4, and -9 were detected with Western blotting and a fluorometric assay. Western blotting was also used to detect the expression of Bcl-2, Bax, cytochrome C, and endoplasmic reticulum (ER) markers.

Results: Emodin (1, 10, and 100 μmol/L) inhibited the growth of human T cells and induced apoptosis in dose- and time dependent manners. Emodin triggered ER stress and significantly elevated intracellular free Ca(2+) in human T cells. It also disrupted mitochondrial membrane potential, and increased cytosolic level of cytochrome C, and the levels of activated cleavage fragments of caspase-3, -4, and -9 in human T cells. Furthermore, emodin significantly increased the levels of ROS and MDA, inhibited both SOD level and GSH/GSSG ratio in human T cells, whereas co-incubation with the ROS scavenger N-acetylcysteine (NAC, 20 μmol/L) almost completely blocked emodin-induced ER stress and mitochondrial dysfunction in human T cells, and decreased the caspase cascade-mediated apoptosis.

Conclusion: Emodin exerts immunosuppressive actions at least partly by inducing apoptosis of human T cells, which is triggered by ROS-mediated ER stress and mitochondrial dysfunction.

Figure 1

Effect of emodin treatment on the growth of human T cells. (A) Morphology changes of human T cells after treatment with emodin at different concentrations (1, 10, or 100 μmol/L). (B) The inhibition of T cell growth induced by Emodin was detected by MTT assay. Data are represented as mean±SD from three independent experiments.

Figure 2

Emodin induced apoptosis and DNA damage of human T cells. (A) and (C) AO/EB double staining was used to detect cell apoptosis after emodin treatment at different concentrations (1, 10, or 100 μmol/L). (B) and (D) Emodin-induced apoptosis of human T cells was detected by Annexin V/PI staining. (E) and (F) Comet assay was used to detect DNA damage. The results are given as mean±SD from three experiments. ^bP<0.05, ^cP<0.01 vs control groups.

Figure 3

Expression and activity of caspases in human T cells after emodin treatment. (A) Pro- and cleaved caspase-9, -4, and -3 of human T cells after emodin treatment with different doses were detected by Western blotting. (B) Pro- and cleaved caspase-8 were detected by Western blotting. (C), (D) and (E) The results of Western blotting were analyzed by Quantity One software. (F), (G), and (H) Activity of caspase-9, -4, and -3 were detected by caspase activity assay. The results are given as mean±SD from three experiments. ^bP<0.05, ^cP<0.01 vs control.

Figure 4

Endoplasmic reticulum stress and intracellular free calcium elevation of human T cells after emodin treatment. (A) Markers of ER stress including GRP78, ATF4, XBP1s and CHOP were detected by Western blotting. (B) The results of Western blotting were analyzed by Quantity One software. (C) Intracellular calcium measurement shown by Fluo-3-AM staining after emodin treatment for 72 h. (D) Positive rate of Fluo-3-AM staining in different emodin treatment groups. The results are given as mean±SD from three experiments. ^bP<0.05, ^cP<0.01 vs control.

Figure 5

The disrupted mitochondrial membrane potential and Cyt c release of human T cells after emodin treatment. (A) JC-1 staining was used to detect ΔΨm level in human T cells treated by 100 μmol/L emodin for 72 h. (B) Analysis of the ΔΨm change in different comcentrations emodin treated T cells. (C) and (D) Alteration of Bcl-2 and Bax in T cells after 100 μmol/L emodin treatment for 24–72 h. (E) and (F) Cyt c release from mitochondria to cytosol in T cells after 100 μmol/L emodin treatment for 24–72 h. The results are given as mean±SD from three experiments. ^bP<0.05, ^cP <0.01 vs control groups.

Figure 6

Emodin significantly induced ROS generation in human T cells. (A) DCFH staining was used to detect ROS level in human T cells treated by 100 μmol/L emodin for 72 h. (B) Analysis of ROS changes in different emodin treated T cells. (C) Effect of emodin on intracellular SOD activity. (D) Effect of emodin on intracellular MDA activity. (E) Effect of emodin on intracellular GSH level. (F) Effect of emodin on intracellular GSSG level. (G) Analysis of the GSH/GSSG ratio. The results are given as mean±SD from three experiments. ^bP<0.05, ^cP<0.01 vs control groups.

Figure 7

Effect of NAC-replenishing on emodin-induced ER stress, mitochondrial dysfunction and apoptosis in T cells. Replenishing of 20 μmol/L exogenous NAC significangly reduced the ROS (A) and MDA (C) production induced by 100 μmol/L emodin, and elevated antioxidant SOD level (B). NAC replenishing also rescued ER stress (G and H), Ca²⁺ release (D), disruption of ΔΨm (E), mitochondrial dysfunction and cytochrome c release (I and J) induced by emodin, which finally inactivated caspases (K and L), and reduced apoptosis of T cells (F). The results are given as mean±SD from three experiments. ^bP<0.05, ^cP<0.01 vs control groups.

Figure 8

Overview of the ROS-mediated ER stress and mitochondrial dysfunction in emodin-induced apoptosis in human T cells. Treatment of T cells with emodin results in ER stress by upregulating GRP78, ATF-4, XBP-1s, and CHOP. As the key transcription factors induced by ER stress, they would trigger the Ca²⁺ release. Emodin also reduces the mitochondrial membrane potential and Bcl-2/Bax ratio, contributing to the activation of caspase-4, -9, and -3 and apoptosis progression. The emodin-induced ER stress and mitochondrial dysfunction require the ROS generation, and on the contrary, antioxidant NAC suppresses emodin-induced apoptosis in human T cells.