One compound of saponins from Disocorea zingiberensis protected against experimental acute pancreatitis by preventing mitochondria-mediated necrosis

Abstract

Acute pancreatitis (AP) is a painful inflammatory disorder of the exocrine pancreas, ranking as the most common gastrointestinal reasons for hospitalization with no specific therapy currently. Diosgenyl saponins extracted from natural products and diosgenin or its derivatives have been shown to exert anti-inflammatory effects in various diseases. However, the therapeutic effects of diosgenyl saponins from Dioscorea zingiberensis C. H. Wright in AP have not yet been determined. Five compounds were extracted and screened for taurocholate-induced necrosis in mouse pancreatic acinar cells. Particularly, 26-O-β-d-glucopyranosyl-3β, 22α, 26-trihydroxy-25(R)-furosta-5-en-3-O-[α-L-rhamnopyranosyl-(1 → 4)]-β-d-glucopyranoside (compound 1) exhibited the best protective effects with no toxicity observed. Next, we showed compound 1 concentration-dependently inhibited necrotic cell death pathway activation and 2.5 mM compound 1 also prevented the loss of mitochondrial membrane potential, adenosine triphosphate production, and reactive oxygen species generation in mouse pancreatic acinar cells. Finally, we showed compound 1 protected against three clinically representative murine models of AP and significantly improved pancreatitis-associated acute lung injury. These data provide in vitro and in vivo evidence that one compound of diosgenyl saponins can be potential treatment for AP. This study suggests natural saponins may serve as fruitful sources for exploring/identifying potential therapies for inflammatory diseases.

Figure 1. Structures of compound 1-5 and cytoprotective effects assessed by necrosis in mouse pancreatic acinar cells.

(a) 26-O-β-d-glucopyranosyl-3β, 22α, 26-trihydroxy-25(R)-furosta-5-en-3-O-[α-L-rhamnopyranosyl-(1 → 4)]-β-d-glucopyranoside diosgenin, (b) zingiberensis saponin, (c) deltonin, (d) diosgenin diglucoside, (e) trillin. Necrosis (%) was calculated by propidium iodide (PI) positive staining divided by Hoechst 33342 positive staining × 100. Data were represented as mean ± S.E.M. and n ≥ 3 per condition. ^# and * p < 0.05 vs control and taurocholic acid sodium salt hydrate (NaT), respectively.

Figure 2. Compound 1 concentration-dependently protected against necrotic cell death pathway activation in mouse pancreatic acinar cells.

(a) Representative images showing Hoechst 33342 (blue) and PI (red) staining from control, 5 mM NaT and 5 mM NaT treated with 2.5 mM compound 1. (b) Necrosis (%) was calculated by PI positive staining divided by Hoechst 33342 positive staining × 100. Data were represented as mean ± S.E.M. and n ≥ 3 per condition. ^# and * p < 0.05 vs control and NaT, respectively.

Figure 3. Compound 1 prevented the loss of ΔΨm, ATP depletion and ROS production induced by NaT in mouse pancreatic acinar cells.

(a) Representative images of JC-1 staining from control, NaT and NaT plus 2.5 mM compound 1, JC-1 manifests red fluorescence at high membrane potential while green fluorescence appears at low membrane potential. (b) The changes of ΔΨm represented as the ratio between red and green fluorescence of JC-1. (c) ATP levels were measured by luminescence. Data were normalized to the control as 100%. (d) Typical trace showing the inhibitory effect of compound 1 (2.5 mM) on NaT-induced ROS production (H₂DCFDA, F/F₀). (e) Quantification of ROS production induced by NaT with or without compound 1.Values were represented as mean ± S.E.M. and n ≥ 3 per condition, ^# and * p < 0.05 vs control and NaT, respectively.

Figure 4. Compound 1 markedly reduced biochemical responses in NaT-AP.

(a) Schema for administration of compound 1 after induction of pancreatitis by retrograde pancreatic ductal injection of 3.5% NaT. Retrograde pancreatic ductal injection of 3.5% NaT caused marked elevations of (b) serum amylase, (c) serum lipase, and (d) pancreatic myeloperoxidase (MPO). Intraperitoneal injection of compound 1 at the dose of 10 and 20 mg/kg significantly reduced all parameters with a more pronounced reduction at the high dose. Data were represented as mean ± S.E.M. and n = 6 per group. ^# and * p < 0.05 vs control and NaT, respectively; ^&p < 0.05, low dose vs high dose.

Figure 5. Compound 1 markedly reduces pancreatic histopathology in NaT-AP.

(a) Representative haematoxylinand & eosin (H&E) section of pancreas from control, NaT, NaT treated with 10 mg/kg compound 1, and NaT treated with 20 mg/kg compound 1. (b) Blinded histopathological analysis for edema, inflammation, necrosis and total histological scores. Intraperitoneal injection of compound 1 at the dose of 10 and 20 mg/kg significantly reduced all score with a more marked reduction at the high dose. Data were represented as mean ± S.E.M. and n = 6 per group. ^# and * p < 0.05 vs control and NaT, respectively; ^&p < 0.05, low dose vs high dose.

Figure 6. Compound 1 markedly reduced biochemical and histological pancreatic damage in CER-AP.

(a) Schema for administration of compound 1 in CER-AP. Seven hourly intraperitoneal injection of CER caused a substantial increase of (b) amylase, lipase, pancreatic trypsin activity and pancreatic MPO activity as well as (c) blinded histopathological scores for edema, inflammation, necrosis and total histological scores. Intraperitoneal administration of 10 and 20 mg/kg compound 1 significantly reduced all biochemical and histological parameters. Data were represented as mean ± S.E.M. and n = 6 per group. ^# and * p < 0.05 vs control and CER, respectively; ^&p < 0.05, low dose vs high dose.(d) Representative H&E section of pancreas from control, CER, CER treated with 10 mg/kg compound 1 and CER treated with 20 mg/kg Compound 1.

Figure 7. Compound 1 markedly reduced biochemical and histological pancreatic damage in FAEE-AP.

(a) Schema for administration of compound 1 in FAEE-AP. Two hourly intraperitoneal injection of a mixture of palmitoleic acid and ethanol caused a substantial elevation of (b) amylase, lipase, pancreatic trypsin activity and pancreatic MPO activity as well as (c) blinded histopathological scores for edema, inflammation, necrosis and total histological scores. Intraperitoneal administration of 10 mg/kg compound 1 significantly reduced all biochemical and histological parameters with a trend towards decrease all at 20 mg/kg. Data were represented as mean ± S.E.M. and n = 6 per group. ^# and * p < 0.05 vs control and FAEE, respectively. (d) Representative H&E section of pancreas from control, FAEE, FAEE treated with 10 mg/kg compound 1 and FAEE treated with 20 mg/kg compound 1.

Figure 8. Compound 1 protected against pancreatitis-associated acute lung injury in NaT-AP.

Retrograde pancreatic ductal injection of 3.5% NaT caused a dramatic lung injury assessed biochemically by (a) IL-6 and (b) lung MPO activity. Intraperitoneal administration of compound 1 at the dose of 10 and 20 mg/kg significantly reduced both markers with more pronounced reduction at the high dose. (c) Representative H&E sections of lungs from control, NaT, NaT treated with 10 mg/kg compound 1 and NaT treated with 20 mg/kg compound 1. Lung histopathological analysis was blindly assessed by (d) edema and (e) inflammation. Data were represented as mean ± S.E.M. and n = 6 per group. ^# and * p < 0.05 vs control and NaT, respectively; ^&p < 0.05, low dose vs high dose.