Pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during alcoholic chronic pancreatitis (ACP). Transforming growth factor‐beta1 (TGF‐β1) is a key regulator of extracellular matrix production and PSC activation. Endotoxin lipopolysaccharide (LPS) has been recognized as a trigger factor in the pathogenesis of ACP. This study aimed to investigate the mechanisms by which LPS modulates TGF‐β1 signalling and pancreatic fibrosis. Sprague‐Dawley rats fed with a Lieber‐DeCarli alcohol (ALC) liquid diet for 10 weeks with or without LPS challenge during the last 3 weeks. In vitro studies were performed using rat macrophages (Mφs) and PSCs (RP‐2 cell line). The results showed that repeated LPS challenge resulted in significantly more collagen production and PSC activation compared to rats fed with ALC alone. LPS administration caused overexpression of pancreatic TLR4 or TGF‐β1 which was paralleled by an increased number of TLR4‐positive or TGF‐β1‐positive Mφs or PSCs in ALC‐fed rats. In vitro, TLR4 or TGF‐β1 production in Mφs or RP‐2 cells was up‐regulated by LPS. LPS alone or in combination with TGF‐β1 significantly increased type I collagen and α‐SMA production and Smad2 and 3 phosphorylation in serum‐starved RP‐2 cells. TGF‐β pseudoreceptor BAMBI production was repressed by LPS, which was antagonized by Si‐TLR4 RNA or by inhibitors of MyD88/NF‐kB. Additionally, knockdown of Bambi with Si‐Bambi RNA significantly increased TGF‐β1 signalling in RP‐2 cells. These findings indicate that LPS increases TGF‐β1 production through paracrine and autocrine mechanisms and that LPS enhances TGF‐β1 signalling in PSCs by repressing BAMBI via TLR4/MyD88/NF‐kB activation.

Pancreatic stellate cells (PSCs) play a key role in fibrogenesis during alcoholic chronic pancreatitis (ACP). Transforming growth factor-β1 (TGF-β1) is a major regulator of PSC activation and extracellular matrix production. Interleukin-6 (IL-6) has shown to participate in TGF-β1 production and rat PSC activation. This study aimed to investigate whether IL-6 promotes human PSC activation and collagen 1(Col1) production through the TGF-β1/Smad pathway. Our results showed that the expression of IL-6 and IL-6R in activated PSCs and macrophages (Mφs) were enhanced in the pancreas of ACP compared to healthy controls and that the mRNA expression of IL-6, IL-6R, TGF-β1, α-SMA or Col1a1 were significantly increased in the pancreas of ACP, showing positive correlations between elevated IL-6 levels and either TGF-β1 or α-SMA or Col1a1 levels and between elevated TGF-β1 levels and α-SMA or Col1a1 levels. In in vitro studies, we identified that IL-6R expression or IL-6 and TGF-β1 secretions were significantly increased in, respectively, Mφs and PSCs by ethanol (EtOH) or lipopolysaccharide (LPS) stimulation while EtOH- or LPS-induced α-SMA or Col1a1 mRNA and protein production in PSCs were partially blocked by IL-6 antibody. IL-6-induced TGF-β1 production in PSCs was antagonized by si-IL-6R RNA or by an inhibitor of STAT3. Additionally, IL-6-promoted α-SMA or Col1a1 protein production was blocked by TGF-β1 antibody and IL-6-induced phosphorylation of Smad2/3 and transcription of α-SMA and Col1a1 mRNA were antagonized by si-TGF-β1 RNA. Our findings indicate that IL-6 contributes to PSC activation and Col1 production through up-regulation of TGF-β1/Smad2/3 pathway.

Pancreatic stellate cells (PSCs) are activated following loss of cytoplasmic vitamin A (retinol)-containing lipid droplets, which is a key event in the process of fibrogenesis of chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDCA). PSCs are the major source of cancer-associated fibroblasts (CAFs) that produce stroma to induce PDAC cancer cell growth, invasion, and metastasis. As an active metabolite of retinol, retinoic acid (RA) can regulate target gene expression in PSCs through its nuclear receptor complex (RAR/RXR or RXR/RXR) or transcriptional intermediary factor. Additionally, RA also has extranuclear and non-transcriptional effects. In vitro studies have shown that RA induces PSC deactivation which reduces extracellular matrix production through multiple modes of action, such as inhibiting TβRⅡ, PDGFRβ, β-catenin and Wnt production, downregulating ERK1/2 and JNK phosphorylation and suppressing active TGF-β1 release. RA alone or in combination with other reagents have been demonstrated to have an effective anti-fibrotic effect on cerulein-induced mouse CP models in vivo studies. Clinical trial data have shown that repurposing all-trans retinoic acid (ATRA) as a stromal-targeting agent for human pancreatic cancer is safe and tolerable, suggesting the possibility of using RA for the treatment of CP and PDCA in humans. This review focuses on RA signaling pathways in PSCs and the effects and mechanisms of RA in PSC-mediated fibrogenesis as well as the anti-fibrotic and anti-tumor effects of RA targeting PSCs or CAFs in vitro and in vivo, highlighting the potential therapies of RA against CP and PDAC.

Chronic pancreatitis (CP) is a chronic inflammatory and fibrotic disease of the pancreas. The incidence of CP is increasing worldwide but the effective therapies are lacking. Hence, it is necessary to identify economical and effective agents for the treatment of CP patients. Vitamin D (VD) and its analogues have been confirmed as pleiotropic regulators of cell proliferation, apoptosis, differentiation and autophagy. Clinical studies show that VD deficiency is prevalent in CP patients. However, the correlation between VD level and the risk of CP remains controversial. VD and its analogues have been demonstrated to inhibit pancreatic fibrosis by suppressing the activation of pancreatic stellate cells and the production of extracellular matrix. Limited clinical trials have shown that the supplement of VD can improve VD deficiency in patients with CP, suggesting a potential therapeutic value of VD in CP. However, the mechanisms by which VD and its analogues inhibit pancreatic fibrosis have not been fully elucidated. We are reviewing the current literature concerning the risk factors for developing CP, prevalence of VD deficiency in CP, mechanisms of VD action in PSC-mediated fibrogenesis during the development of CP and potential therapeutic applications of VD and its analogues in the treatment of CP.

Human pancreatic stellate cells (PSCs) play a critical role in fibrogenesis during chronic pancreatitis (CP). However, primary PSCs have a short lifespan in vitro, which seriously affects their use in various applications. We have established a stable immortalized human PSC line (HP-1) by RSV promoter/enhancer-driven SV40 T antigen expression in primary activated human PSCs. HP-1 cells express cytoskeleton proteins including glial fibrillary acidic protein (GFAP), α-smooth muscle actin (α-SMA), vimentin and desmin, and are typical of PSCs, which are high transfeciability and viable in 0.5% serum. The cells express receptors such as TGFβR2, PDGFR, TGF-β pseudoreceptor Bambi and PPRPγ that are commonly found in PSCs. HP-1 cells are similar to activated human PSCs in that they have enhanced expression of α-SMA, CTGF, Col1 and TIMP-2 mRNAs or proteins, as well as decreased expression of MMP-1/2 mRNAs or proteins in response to TGF-β1 stimulation. Comparative proteomics revealed 4,537 shared proteins between HP-1 cells and PSCs and no single protein in HP-1 cells versus PSCs. Statistical analysis reveals no significantly difference between HP-1 cells and PSCs in their expression of proteins associated with matrix and matrix remodeling. The similarity between HP-1 cell and PSC is further shown by the finding that only 9 proteins are differentially up-regulated > ± 2-fold in HP-1 cells and 13 proteins are up-regulated > ± 2-fold in PSCs and none of these proteins include ECM proteins, cytokines, growth factors or matrix remodeling regulatory proteins. Therefore, HP-1 cells can be used as an effective tool for the study of PSC-mediated pancreatic fibrosis.

To evaluate the inhibitory effect of vitamin D₃ analogue calcipotriol (Cal) on the fibrosis of pancreatic stellate cells (PSCs) induced by TGF-β1 and the rationality of Cal use in alcoholic chronic pancreatitis (ACP). Double-labeling immunofluorescence was used for the identification of VDR⁺PSCs in the pancreas of healthy controls (HC) and ACP patients. Van Gieson staining for examination of collagen fibers. RT-qPCR and Western Blot for determining the mRNAs and proteins of VDR, TGF-β1 and COL1A1 in the pancreas of ACP or in vitro PSCs. ELISA or LC-MS/MS for detection of serum TGF-β1 and COL1A1 or 25(OH)D_3. The PSC line (RP-2 cell) was used for the determination of proteomic alterations in Cal plus TGF-β1 versus TGF-β1 and to examine the effect of VDR gene knockdown. Enhanced expression of VDR was detected in RP-2 cells stimulated with alcohol (ALC) plus Cal versus Cal alone and in PSCs in the pancreas of ACP versus HC. The increased VDR⁺PSCs were positively correlated with the levels of COL1A1 mRNAs or areas of collagen deposition in the pancreas of ACP. TGF-β1 was overexpressed in the pancreas of ACP and ALC-treated RP-2 cells while 25(OH)D₃ level in serum was significantly decreased in ACP versus HC. Through a VDR-dependent mechanism, Cal antagonized 16 profibrotic proteins in TGF-β1-induced RP-2 cells that included 7 extracellular matrix components, 2 cytoskeletal proteins, 2 fibrosis-associated factors (RUNX1 and TRAF2), TIMP-1, CCN1, integrin α11, an adhesion scaffold protein (TGFB1i1) and an enzyme mediating TGF-β1-induced fibrogenesis (ENPP1). This study suggests that Cal administration may be a potential antifibrotic strategy via inhibiting TGF-β1-mediated PSC action during the development of ACP.