Dihydrodiosgenin protects against experimental acute pancreatitis and associated lung injury through mitochondrial protection and PI3Kγ/Akt inhibition

Abstract

Background and purpose: Acute pancreatitis (AP) is a painful and distressing disorder of the exocrine pancreas with no specific treatment. Diosgenyl saponins extracted from from Dioscorea zingiberensis C. H. Wright have been reported to protect against experimental models of AP. Diosgenin, or its derivatives are anti-inflammatory in various conditions. However, the effects of diosgenin and its spiroacetal ring opened analogue, dihydrodiosgenin (Dydio), on AP have not been determined.

Experimental approach: Effects of diosgenin and Dydio on sodium taurocholate hydrate (Tauro)-induced necrosis were tested, using freshly isolated murine pancreatic acinar cells. Effects of Dydio on mitochondrial dysfunction in response to Tauro, cholecystokinin-8 and palmitoleic acid ethyl ester were also assessed. Dydio (5 or 10 mg·kg^-1 ) was administered after the induction in vivo of Tauro-induced AP (Wistar rats), caerulein-induced AP and palmitoleic acid plus ethanol-induced AP (Balb/c mice). Pancreatitis was assessed biochemically and histologically. Activation of pancreatic PI3Kγ/Akt was measured by immunoblotting.

Key results: Dydio inhibited Tauro-induced activation of the necrotic cell death pathway and prevented pancreatitis stimuli-induced mitochondrial dysfunction. Therapeutic administration of Dydio ameliorated biochemical and histopathological responses in all three models of AP through pancreatic mitochondrial protection and PI3Kγ/Akt inactivation. Moreover, Dydio improved pancreatitis-associated acute lung injury through preventing excessive inflammatory responses.

Conclusion and implications: These data provide in vitro and in vivo mechanistic evidence that the diosgenin analogue, Dydio could be potential treatment for AP. Further medicinal optimization of diosgenin and its analogue might be a useful strategy for identifying lead candidates for inflammatory diseases.

Figure 1

Diosgenin and Dydio protect against Tauro‐induced activation of the necrotic cell death pathway in pancreatic acinar cells isolated from Balb/c mice. Diosgenin (A) and Dydio (B) protect against activation of the necrotic cell death pathway (shown by PI uptake) induced by Tauro (5 mmol·L⁻¹) in isolated mouse pancreatic acinar cells. (C) Representative images showing Hoechst 33342 (blue) and PI (red) staining in pancreatic acinar cells stimulated with Tauro (5 mmol·L⁻¹) in the absence or presence of Dydio (100 μmol·L⁻¹). Data are expressed as means ± SEM; n = 5 experiments per group. ^# P < 0.05, significantly different from control; *P < 0.05, significantly different from Tauro.

Figure 2

Dydio prevents pancreatitis stimuli‐induced mitochondrial dysfunction and Tauro‐induced activation of PI3Kγ/Akt. (A) Representative images of JC‐1 staining (left panel) and quantification of red/green fluorescence intensity (right panel), showing the prevention by Dydio (100 μmol·L⁻¹) of Tauro (5 mmol·L⁻¹), CCK (100 nmol·L⁻¹) or POAEE (50 μmol·L⁻¹)‐induced dissipation of ΔΨm. (B) ATP levels were measured by luminescence in pancreatic acinar cells treated with Tauro (5 mmol·L⁻¹), CCK (100 nmol·L⁻¹) or POAEE (50 μmol·L⁻¹) alone or in combination with Dydio (100 μmol·L⁻¹) for 30 min. Data were normalized to untreated control as 100%. (C) Typical trace showing the inhibitory effect of Dydio (100 μmol·L⁻¹) on Tauro‐induced cytosolic ROS production ([ROS]_i). The effects were quantified at 1500 s and expressed as means ± SEM; n = 5 per condition. ^# P < 0.05, significantly different from control; *P < 0.05, significantly different from Tauro or CCK or POAEE. (D) Traces showing the protective effect of Dydio (1 μmol·L⁻¹) on mitochondrial swelling measured by absorbance at 540 nm. The data are expressed as means ± SEM; n = 5 per condition. (E) Immunoblots for PI3K p100γ and phospho‐Akt (pAkt) expression in freshly isolated mouse pancreatic acinar cells, which were pretreated with 100 μmol·L⁻¹ Dydio and stimulated with 5 mmol·L⁻¹ Tauro for 30 min or 1 h. Data are expressed as means ± SEM; n = 5 per condition. ^# P < 0.05, significantly different from control, *P < 0.05, significantly different from Tauro at 30 min, ^& P < 0.05, significantly different from Tauro at 1 h. (F) 740 Y‐P, a PI3K activator, abolished the protective effect of Dydio on Tauro‐induced necrosis. Cells were pretreated with or without 740 Y‐P (20 μmol·L⁻¹) ± Dydio (100 μmol·L⁻¹) for 30 min and stimulated with 5 mmol·L⁻¹ Tauro for 50 min. Results are expressed as means ± SEM; n = 5 per condition. ^# P < 0.05, significantly different from control; *P < 0.05, significantly different from Tauro.

Figure 3

Dydio attenuates biochemical and histological responses in Tauro‐AP model in vivo. (A) Scheme for administration of Dydio after induction of pancreatitis by retrograde pancreatic ductal injection of 3.5% Tauro. (B) Serum amylase activity, serum lipase activity, serum IL‐6 and pancreatic MPO activity at 24 h after the induction of Tauro‐AP. (C) Representative H&E images of pancreatic sections in Tauro‐AP. (D) Histopathological scores for oedema, inflammation, necrosis and the total histology scores. Injection (i.p.) of Dydio (5 and 10 mg·kg⁻¹) ameliorated biochemical responses and pancreatic histological scores. Data are expressed as means ± SEM; n = 6 rats per group. ^# P < 0.05, significantly different from sham control; *P < 0.05, significantly different from Tauro.

Figure 4

Dydio attenuates biochemical and histological responses of hyperstimulation pancreatitis, with caerulin (CER‐AP). (A) Scheme for administration of Dydio in CER‐AP. (B) Serum amylase activity, serum lipase activity, serum IL‐6, pancreatic trypsin activity and pancreatic MPO activity at 12 h after the first injection of CER. (C) Representative H&E images of the pancreatic sections. (D) Histopathological scores for oedema, inflammation, necrosis and total histological scores. (E) Representative images of the pancreatic sections stained with F4/80 for assessing infiltration of macrophages. Data are expressed as means ± SEM, n = 6 mice per group. ^# P < 0.05, significantly different from control; *P < 0.05, significantly different from CER.

Figure 5

Dydio attenuates biochemical and histological responses of FAEE‐AP. (A) Scheme for administration of Dydio in FAEE‐AP. (B) Serum amylase activity, serum lipase activity, serum IL‐6, pancreatic trypsin activity and pancreatic MPO activity at 24 h after FAEE‐AP induction. (C) Representative H&E images of pancreatic sections. (D) Histopathological scores for oedema, inflammation, necrosis and total histological scores. (E) Representative images of the pancreatic sections stained with F4/80 for assessing the infiltration of macrophages. Data are expressed as means ± SEM, n = 6 mice per group. ^# P < 0.05, significantly different from control; *P < 0.05, significantly different from FAEE.

Figure 6

Dydio prevents activation of pancreatic PI3Kγ/Akt in vivo. (A) PI3K p110γ levels in pancreas from indicated groups were analysed by immunohistochemistry. (B) Immunoblots for PI3K p110γ and pAkt in the pancreas from Tauro‐AP (left), CER‐AP (middle) or FAEE‐AP (right) and the quantification (means ± SEM, n = 5). ^# P < 0.05, significantly different from control; *P < 0.05, significantly different from Tauro, CER or FAEE.

Figure 7

Dydio reduces pancreatitis‐associated acute lung injury. (A) Lung MPO activities in three models. (B) Representative H&E images (200×) of lung tissue from control, Tauro‐AP, Tauro‐AP plus 5 mg·kg⁻¹ Dydio and Tauro‐AP plus 10 mg·kg⁻¹. (C) Histopathological scores for oedema and inflammation. (D) Representative images of immunohistochemical staining for PI3K p110γ from control, Tauro‐AP, Tauro‐AP plus 5 mg·kg⁻¹ Dydio and Tauro‐AP plus 10 mg·kg⁻¹. Data are expressed as means ± SEM, n = 6 per group, ^# P < 0.05, significantly different from sham control, *P < 0.05, significantly different from Tauro‐AP.