Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 20 (4), 3709-3718

Piperine Ameliorates the Severity of Fibrosis via Inhibition of TGF‑β/SMAD Signaling in a Mouse Model of Chronic Pancreatitis

Affiliations

Piperine Ameliorates the Severity of Fibrosis via Inhibition of TGF‑β/SMAD Signaling in a Mouse Model of Chronic Pancreatitis

Ji-Won Choi et al. Mol Med Rep.

Abstract

Chronic pancreatitis (CP) is characterized by recurrent pancreatic injury, resulting in inflammation and fibrosis. Currently, there are no drugs for the treatment of pancreatic fibrosis associated with CP. Piperine, a natural alkaloid found in black pepper, has been reported to show anti‑inflammatory, anti‑oxidative, and antitumor activities. Although piperine exhibits numerous properties in regards to the regulation of diverse diseases, the effects of piperine on CP have not been established. To investigate the effects of piperine on CP in vivo, we induced CP in mice through the repetitive administration of cerulein (50 µg/kg) six times at 1‑h intervals, 5 times per week, for a total of 3 weeks. In the pre‑treatment groups, piperine (1, 5, or 10 mg/kg) or corn oil were administrated orally at 1 h before the first cerulein injection, once a day, 5 times a week, for a total of 3 weeks. In the post‑treatment groups, piperine (10 mg/kg) or corn oil was administered orally at 1 or 2 week after the first cerulein injection. Pancreases were collected for histological analysis. In addition, pancreatic stellate cells (PSCs) were isolated to examine the anti‑fibrogenic effects and regulatory mechanisms of piperine. Piperine treatment significantly inhibited histological damage in the pancreas, increased the pancreatic acinar cell survival, reduced collagen deposition and reduced pro‑inflammatory cytokines and chemokines. In addition, piperine treatment reduced the expression of fibrotic mediators, such as α‑smooth muscle actin (α‑SMA), collagen, and fibronectin 1 in the pancreas and PSCs. Moreover, piperine treatment reduced the production of transforming growth factor (TGF)‑β in the pancreas and PSCs. Furthermore, piperine treatment inhibited TGF‑β‑induced pSMAD2/3 activation but not pSMAD1/5 in the PSCs. These findings suggest that piperine treatment ameliorates pancreatic fibrosis by inhibiting TGF‑β/SMAD2/3 signaling during CP.

Figures

Figure 1.
Figure 1.
Effects of piperine on pancreatic damage caused by cerulein-induced CP in mice. (A) CP was induced by the administration of six intraperitoneal injections of cerulein (50 µg/kg) at 1-h intervals, 5 days per week, for a total of 3 weeks. Piperine or corn oil was administrated orally at 1 h before the first cerulein injection, once a day, 5 times a week for a total of 3 weeks. The groups of mice were euthanized 3 weeks after the beginning of the cerulein treatment. (B) Representative H&E-stained pancreatic tissue sections of the control mice and piperine-treated mice (1, 5 or 10 mg/kg) before the first cerulein (50 µg/kg)-mediated induction of CP. (C) Histological sections of the pancreas were scored from 0 (normal) to 3 (severe) for inflammation and glandular atrophy. Data are represented as means ± SEM for 6 mice in each group. Results are representative of three experiments. *P<0.05 vs. corn oil alone; P<0.05 vs. CP. Original magnification, ×200. CP, chronic pancreatitis; H&E, hematoxylin and eosin.
Figure 2.
Figure 2.
Effect of piperine on PSC activation during CP. (A) Confocal images of immunofluorescence staining of α-SMA (red) and DAPI (blue). (B) Relative intensity of α-SMA. (C) Acta-2 mRNA was assessed using RT-qPCR. Data are represented as means ± SEM for 6 mice in each group. Results are representative of three experiments. *P<0.05 vs. corn oil alone; P<0.05 vs. CP. Scale bar, 20 µm. PSC, pancreatic stellate cell; CP, chronic pancreatitis; α-SMA, α-smooth muscle actin; DAPI, 4′,6-diamidino-2-phenylindole.
Figure 3.
Figure 3.
Effect of piperine on ECM deposition during CP. (A) Sirius red staining for collagen deposition. (B) Relative intensity of Sirius Red. The pancreatic mRNA levels of (C) collagen I and (D) fibronectin 1 were assessed using RT-qPCR. Data are represented as means ± SEM for 6 mice in each group. Results are representative of three experiments. *P<0.05 vs. corn oil alone; P<0.05 vs. CP. Original magnification, ×200. CP, chronic pancreatitis; ECM, extracellular matrix.
Figure 4.
Figure 4.
Effect of piperine on cytokines (TGF-β, TNF-α, IL-1β and IL-6 mRNA) and chemokines (CCL2 and CXCL2 mRNA) production during CP. (A) Confocal images of immunofluorescence staining of TGF-β (green) and DAPI (blue). (B) Relative intensity of TGF-β. (C) TGF-β mRNA was determined using RT-qPCR. (C) TGF-β, (D) TNF-α, IL-1β and IL-6, (E) CCL2 and CXCL2 mRNA was determined using RT-qPCR. Data are represented as means ± SEM for 6 mice in each group. Results are representative of three experiments. *P<0.05 vs. corn oil alone; P<0.05 vs. CP. Scale bar, 10 µm. CCL, C-C motif chemokine ligand; CXCL, C-X-C motif chemokine ligand; CP, chronic pancreatitis; IL, interleukin; TGF-β, transforming growth factor-β; TNF, tumor necrosis factor; DAPI, 4′,6-diamidino-2-phenylindole.
Figure 5.
Figure 5.
Effect of piperine on TGF-β-induced PSC activation and ECM production on the isolated PSCs. Mouse PSCs were pretreated with piperine at various concentrations (10, 20, 50 µM) for 1 h, and then stimulated with TGF-β (0.5 ng/ml) for 24 h. Levels of mRNA were quantified by RT-PCR for (A) α-SMA, (B) fibronectin 1, (C) collagen I, (D) collagen III, (F) TGF-β. (E) Protein levels of α-SMA and collagen I were assessed using western blot analysis and GAPDH was used as a loading control. Mouse PSCs were pretreated with piperine (50 µM) for 1 h followed by TGF-β (0.5 ng/ml) for 30 min, and then whole cell lysates were harvested. (G) The phosphorylation (p) levels of SMAD2/3 and SMAD 1/5 were analyzed by western blot analysis and SMAD3 and SMAD5 were used as loading controls. Data are represented as means ± SEM for 6 mice in each group. Results are representative of three experiments. *P<0.05 vs. saline alone; P<0.05 vs. TGF-β. TGF-β, transforming growth factor-β; PSCs, pancreatic stellate cells; ECM, extracellular matrix; CP, chronic pancreatitis; α-SMA, α-smooth muscle actin.
Figure 6.
Figure 6.
Therapeutic effects of piperine on cerulein-induced CP in mice. (A) CP was induced by the administration of six intraperitoneal injections of cerulein (50 µg/kg) at 1-h intervals, 5 days per week, for a total of 3 weeks. Piperine or corn oil was administrated orally 1 or 2 week after the onset of the first cerulein injection, once a day, 5 days per week, for a total of 2 or 1 week. Groups of mice were euthanized 3 weeks after the beginning of the cerulein treatment. (B) Representative H&E-stained pancreatic tissue sections. (C) Histological sections of the pancreas were scored from 0 (normal) to 3 (severe) for glandular atrophy and inflammation. (D) Immunofluorescence staining of α-SMA (red) and DAPI (blue). (E) Relative intensity of α-SMA. (F) Sirius red staining. (G) Relative intensity of Sirius Red. The pancreatic mRNA levels of (H) Acta-2, (I) collagen I, (J) fibronectin 1 and (K) TGF-β were determined using RT-qPCR. Data are represented as means ± SEM for 6 mice in each group. Results are representative of three experiments. *P<0.05 vs. corn oil alone; P<0.05 vs. CP. Original magnification, ×200. Scale bar, 20 µm. CP, chronic pancreatitis; H&E, hematoxylin and eosin; α-SMA, α-smooth muscle actin; TGF-β, transforming growth factor-β; DAPI, 4′,6-diamidino-2-phenylindol.

Similar articles

See all similar articles

Cited by 1 PubMed Central articles

References

    1. Apte M, Pirola R, Wilson J. The fibrosis of chronic pancreatitis: New insights into the role of pancreatic stellate cells. Antioxid Redox Signal. 2011;15:2711–2722. doi: 10.1089/ars.2011.4079. - DOI - PubMed
    1. Hobbs PM, Johnson WG, Graham DY. Management of pain in chronic pancreatitis with emphasis on exogenous pancreatic enzymes. World J Gastrointest Pharmacol Ther. 2016;7:370–386. doi: 10.4292/wjgpt.v7.i3.370. - DOI - PMC - PubMed
    1. Yang AL, Vadhavkar S, Singh G, Omary MB. Epidemiology of alcohol-related liver and pancreatic disease in the United States. Arch Intern Med. 2008;168:649–656. doi: 10.1001/archinte.168.6.649. - DOI - PubMed
    1. Robles-Diaz G, Vargas F, Uscanga L, Fernández-del Castillo C. Chronic pancreatitis in Mexico City. Pancreas. 1990;5:479–483. doi: 10.1097/00006676-199007000-00017. - DOI - PubMed
    1. Hirota M, Shimosegawa T, Masamune A, Kikuta K, Kume K, Hamada S, Kihara Y, Satoh A, Kimura K, Tsuji I, et al. The sixth nationwide epidemiological survey of chronic pancreatitis in Japan. Pancreatology. 2012;12:79–84. doi: 10.1016/j.pan.2012.02.005. - DOI - PubMed
Feedback