Chronic pancreatitis (CP) is a debilitating inflammatory disease of the exocrine pancreas, increasing pancreatic cancer risk. CP is characterized by acinar cell injury and progressive pancreatic fibrosis, leading to functional loss. Currently, there's no effective therapy to reverse fibrotic damage. Activated pancreatic stellate cells (PSCs) are central to extracellular matrix (ECM) formation in pancreatic fibrosis. Normally quiescent and vitamin A-containing, PSCs become activated in response to injury or inflammation, exhibiting increased α-smooth muscle actin (α-SMA) expression and ECM protein release. Transforming growth factor β (TGF-β) is a major PSC activator, triggering TGF-β1-dependent Smad2 signaling. PSCs share similarities with hepatic stellate cells (HSCs). Previous research showed hydrogen peroxide-inducible clone 5 (Hic-5) plays a crucial role in liver fibrosis, with Hic-5 deficient mice exhibiting reduced liver fibrosis and HSC activation. Hic-5, also known as TGF-β-1-induced transcript 1 (TGFβI11), is an adhesion scaffold protein affecting the cytoskeleton and essential for ECM deposition and remodeling. Its role in CP, however, was unknown. This study investigated Hic-5's role in PSC activation and pancreatic fibrosis in CP, hypothesizing that Hic-5 is involved in PSC activation and subsequent fibrosis development in CP.

Existing literature strongly implicates activated PSCs in the pathogenesis of pancreatic fibrosis in chronic pancreatitis. Studies have demonstrated the crucial role of TGF-β signaling, particularly the Smad2 pathway, in PSC activation and ECM production. The similarities between PSCs and hepatic stellate cells (HSCs) suggest potential parallels in the molecular mechanisms driving their activation. Previous work on Hic-5 in liver fibrosis established its role in HSC activation and ECM deposition through regulation of TGF-β/Smad signaling. However, the specific role of Hic-5 in the context of pancreatic fibrosis and CP remained unexplored prior to this study.

The study used both in vivo and in vitro approaches. For the in vivo component, wild-type (WT) and Hic-5 knockout (Hic-5 KO) mice were subjected to caerulein injections to induce CP. The severity of CP was assessed by pancreatic weight, histological analysis (H&E, Sirius Red, Masson's trichrome staining), and Western blotting and qPCR for α-SMA and collagen expression. For the in vitro component, PSCs were isolated from WT and Hic-5 KO mice and cultured. The activation state of PSCs was evaluated using immunofluorescence (α-SMA and Hic-5), Western blotting, and qPCR. The effect of Hic-5 deficiency on TGF-β/Smad2 signaling was investigated by measuring phosphorylated Smad2 levels. In addition, human primary PSCs were obtained commercially and subjected to Hic-5 knockdown using siRNA, followed by similar analyses as in the mouse PSC experiments. Statistical analysis involved Student's t-test.

The study revealed significantly enhanced Hic-5 expression in activated PSCs from both human CP tissue and the caerulein-induced mouse CP model. Hic-5 deficiency in mice significantly attenuated pancreatic fibrosis and PSC activation. Histological analysis showed a marked reduction in acinar cell loss and fibrosis in Hic-5 KO mice compared to WT mice. Western blot and qPCR analyses confirmed reduced α-SMA and collagen I/III expression in Hic-5 KO mice. In vitro studies showed that Hic-5 KO PSCs exhibited significantly reduced α-SMA and collagen I expression compared to WT PSCs. Importantly, Hic-5 deficiency impaired Smad2 phosphorylation in cultured murine PSCs, indicating a role for Hic-5 in TGF-β/Smad signaling. In human PSCs, Hic-5 knockdown using siRNA also reduced α-SMA and collagen I expression and impaired Smad2 phosphorylation. These results suggest Hic-5 plays a critical role in mediating TGF-β/Smad signaling in PSC activation and fibrosis development during CP. There was no significant difference in TGF-β1 mRNA expression levels between WT and Hic-5 KO mice in either the in vivo or in vitro studies.

This study demonstrates that Hic-5 plays a critical role in the development of pancreatic fibrosis in CP by mediating the activation of PSCs. The significant reduction in pancreatic fibrosis and PSC activation in Hic-5 KO mice, coupled with the in vitro findings showing impaired Smad2 phosphorylation in Hic-5 deficient PSCs, strongly supports this conclusion. The findings highlight Hic-5 as a potential therapeutic target for CP. Targeting Hic-5 could offer a novel approach to mitigate the fibrotic process and improve outcomes in CP patients. This research advances the understanding of PSC activation mechanisms, offering a potential therapeutic strategy focused on inhibiting the Hic-5-mediated TGF-β/Smad2 pathway. The lack of difference in TGF-β1 levels between WT and Hic-5 KO suggests that Hic-5 acts downstream of TGF-β1 in this pathway.

This study shows Hic-5 is significantly upregulated in activated PSCs during CP and plays a crucial role in pancreatic fibrosis development by promoting Smad2 phosphorylation and subsequent collagen production. Hic-5 deficiency, both in vivo and in vitro, attenuates PSC activation and reduces fibrosis. These findings suggest Hic-5 is a promising therapeutic target for CP. Future studies should focus on developing targeted therapies against Hic-5 for CP treatment and exploring its role in other fibrotic diseases.

The study primarily used a caerulein-induced mouse model of CP, which may not perfectly replicate the complexities of human CP. Further investigation in other CP models and human samples is warranted. The in vitro studies used cultured PSCs, which might not fully recapitulate the in vivo microenvironment. While the study focused on the TGF-β/Smad2 pathway, other signaling pathways may also be involved in Hic-5’s effects on PSC activation. The study did not investigate the long-term effects of Hic-5 deficiency on CP progression or potential side effects of Hic-5 inhibition.