Triptolide Suppresses NF-κB-Mediated Inflammatory Responses and Activates Expression of Nrf2-Mediated Antioxidant Genes to Alleviate Caerulein-Induced Acute Pancreatitis

Abstract

Triptolide (TP), the main active ingredient of Tripterygium wilfordii Hook.f., displays potent anti-inflammatory, antioxidant, and antiproliferative activities. In the present study, the effect of TP on acute pancreatitis and the underlying mechanisms of the disease were investigated using a caerulein-induced animal model of acute pancreatitis (AP) and an in vitro cell model. In vivo, pretreatment with TP notably ameliorated pancreatic damage, shown as the improvement in serum amylase and lipase levels and pancreatic morphology. Meanwhile, TP modulated the infiltration of neutrophils and macrophages (Ly6G staining and CD68 staining) and decreased the levels of proinflammatory factors (TNF-α and IL-6) through inhibiting the transactivation of nuclear factor-κB (NF-κB) in caerulein-treated mice. Furthermore, TP reverted changes in oxidative stress markers, including pancreatic glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA), in acute pancreatitis mice. Additionally, TP pretreatment inhibited intracellular reactive oxygen species (ROS) levels via upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) expression and Nrf2-regulated redox genes expression (HO-1, SOD1, GPx1 and NQO1) in vitro. Taken together, our data suggest that TP exert protection against pancreatic inflammation and tissue damage by inhibiting NF-κB transactivation, modulating immune cell responses and activating the Nrf2-mediated antioxidative system, thereby alleviating acute pancreatitis.

Keywords: acute pancreatitis; inflammatory responses; oxidative stress; triptolide.

Figure 1

Triptolide-ameliorated pancreatic damage in mice with caerulein-induced acute pancreatitis. (A) Serum lipase and serum amylase levels; (B) hematoxylin and eosin stained (H&E) section of pancreas. Data shown are means ± SEM. *** p < 0.001, and **** p < 0.0001 compared with control group. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 compared with model group. Triptolide is abbreviated as TP, and tanshinone IIA is abbreviated as TSA.

Figure 2

Triptolide decreased neutrophil infiltration in the mice with caerulein-induced acute pancreatitis. Data shown are means ± SEM. *** p < 0.001 compared with control group. ^### p < 0.001 compared with the model group.

Figure 3

Triptolide decreased macrophage infiltration in mice with caerulein-induced acute pancreatitis. Data shown are means ± SEM. *** p < 0.001 compared with control group. ^## p < 0.01, and ^### p < 0.001 compared with the model group.

Figure 4

Triptolide reduced inflammatory factors in mice with caerulein-induced acute pancreatitis. Serum TNF-α and IL-6 were measured by ELISA. Data shown are means ± SEM. ** p < 0.01 compared with control group. ^# p < 0.05 compared with the model group. Triptolide is represented by TP, and tanshinone IIA is represented by TSA.

Figure 5

Triptolide inhibited NF-κB activation in a caerulein-induced acute pancreatitis model. (A) The expression of pancreatic NF-κB in mice, (B) the expression of NF-κB in 266-6 cells, and (C) immunofluorescence staining of NF-κB in 266-6 cells. Data shown are means ± SEM. * p < 0.05, *** p < 0.001 compared with control group. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 compared with model group. Triptolide is represented by TP, and tanshinone IIA is represented by TSA.

Figure 6

Triptolide alleviates oxidative stress in a caerulein-induced acute pancreatitis model. (A) The levels of ROS and SOD in 266-6 cells, and (B) the levels of SOD, GSH and MDA in pancreatic tissue. Data shown are means ± SEM. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with control group. ^# p < 0.05, ^## p < 0.01, and ^### p < 0.001 compared with model group. Triptolide is represented by TP, and tanshinone IIA is represented by TSA.

Figure 7

Triptolide activated the Nrf2 signaling pathway in a caerulein-induced acute pancreatitis model. (A) The mRNA expression of HO-1, SOD1, GPx1 and NQO1 in 266-6 cells. (B) The mRNA and protein expression of Nrf2 in 266-6 cells. Data shown are means ± SEM. * p < 0.05, ** p < 0.01 compared with control group. ^# p < 0.05, and ^## p < 0.01 compared with the model group. Triptolide is represented by TP, and tanshinone IIA is represented by TSA.