A. A. Mrazek1, V. Bhatia2, M. Falzon2, M. R. Hellmich1, C. Chao1 1University Of Texas Medical Branch,Surgery,Galveston, TX, USA 2University Of Texas Medical Branch,Pharmacology,Galveston, TX, USA
Introduction: Chronic pancreatitis (CP) is characterized by repeated bouts of acute pancreatitis, resulting in inflammation, glandular necrosis, and irreversible stromal fibrosis. Apigenin (Api) is a natural compound which we have shown to preserve acini and significantly inhibit interstitial fibrosis in a preclinical mouse model of CP. We found that Api down-regulates parathyroid hormone related protein (PTHrP), a pro-inflammatory mediator responsible for amplifying acinar and pancreatic stellate cell (PSC) response to injury. In vitro, Api reduced the viability and induced apoptosis PSCs, the cells responsible for secretion of inflammatory cytokines and the over-deposition of extracellular matrix (ECM). The aim of this study is to determine if Api acts at a transcriptional level, limiting the synthesis of inflammatory mediators (IL-6 and IL-8), markers of PSC proliferation (TGF-β and PCNA), and major components of the ECM, collagen type I and fibronectin (COL1A1 and FN).
Methods: Following an IRB-approved protocol, human PSC were isolated from discarded OR tissue by a standard outgrowth method. The PSC were pretreated with Api 50μM for 1h or vehicle (DMSO) and then stimulated with PTHrP (Bachem, Torrance, CA) 1ng/ml for 12h. Total RNA was isolated (Ambion, Austin, TX); RT-PCR was performed using SYBR Green Supermix (Applied Biosystems, Carlsbad, CA). The relative expression of transcripts was normalized to 18S expression. One-way ANOVA and post-hoc Tukey’s test were assessed with SPSS (IBM, Armonk, NY), and significance was set at p<0.05.
Results: Pretreatment with Api significantly reduced basal mRNA expression of IL-6 and IL-8, TGF-β, PCNA, and fibrillar protein COL1A1 (p<0.001) [Table 1], but not FN. When PSCs were stimulated PTHrP, Api significantly decreased the transcriptional response of all transcripts evaluated (p<0.001).
Conclusions: Api significantly lowers the threshold of basal transcriptional activity in PSC of IL-6 and IL-8 (pro-inflammatory cytokines), TGF-β and PCNA (measures of cellular proliferation), and COL1A1 (the most abundant ECM protein). Additionally, Api reduced PTHrP-stimulated increases in all factors evaluated. Pretreatment with Api did not decrease the basal mRNA levels of FN at 12h, an ECM protein which has been shown to be overabundant in CP. Changes in FN mRNA may be revealed at longer time intervals. These RT-PCR results offer mechanistic insight into the action of Api and confirm the preclinical findings in our murine model of CP, where Api reduced pancreatic fibrosis while preserving normal acinar unit architecture. This further supports apigenin’s development as a therapeutic in pancreatitis.
